Sublethal effects of environmental concentrations of caffeine on a neotropical freshwater fish.

Caffeine is a contaminant frequently detected in water bodies. Growth trends in both human population and caffeine consumption per capita are expected to exacerbate the occurrence of caffeine in freshwaters. Yet the effects of caffeine on native fish fauna are poorly understood. We exposed larvae of an endemic Neotropical catfish (Rhamdia quelen) to a range of caffeine concentrations for 30 days. We found that larvae exposed to the highest concentration (16 mg L-1) showed skeletal deformations and reduced growth. We further compiled measured environmental concentrations of caffeine in surface freshwater globally and performed a risk assessment. Our analysis points to a low risk to R. quelen and equally sensitive fish species in ~90% of the freshwater ecosystems considered in our analysis. The risk quotient is higher in freshwater ecosystems of South and Central America, where R. quelen is endemic. Although the ecotoxicological risk is currently low in most places, increased caffeine consumption, exacerbated by the lack of sanitation, is expected to increase caffeine concentrations in many parts of the world, posing a threat of sublethal morphological effects to local fish species.

Assessing the short-term response of fish assemblages to damming of an Amazonian river.

The enormous biodiversity of tropical freshwater combined with a considerable increase in the construction of reservoirs urges to understand the ecological effects caused by damming. Using rarely available data obtained before (one year) and after (four years) the filling of a hydroelectric plant on the Teles Pires River (Amazon River basin), the effects on abundance, biomass, and diversity of the fish assemblage were evaluated using two complementary approaches: a BACI (before-after-control-impact) design with mixed models and analyses of covariance. Significant Before-After × Control-Impact interactions in abundance, biomass, and species richness were observed, with decreases of abundance and species richness and more stable biomass after filling. Some abundant species, such as Jupiaba polylepis, Jupiaba acanthogaster, Knodus cf. heteresthes, and Moenkhausia lepidura among others, declined in abundance or disappeared from the impact sites. However, temporal and particularly spatial variation independent of damming explained more variation in all the response variables analyzed, including species composition, and analyses of covariance demonstrated general negative trends irrespective of damming. This study illustrates the usefulness of BACI designs to assess the effects of damming but also that other statistical approaches are complementary, given the difficulty of identifying control sites and the short length of most ecological time series. The results also suggest that preserving tributaries upstream of reservoirs and natural regimes of spatial and temporal environmental variation might help to mitigate the impacts of damming in tropical ecosystems.

Temporal and Spatial Variability of Micropollutants in a Brazilian Urban River.

In Brazil, environmental occurrence of micropollutants, such as pharmaceuticals, is rarely studied, and these compounds are not part of national water quality guidelines. In this study, we evaluated the occurrence of micropollutants in the Paraibuna River, located in the southeast region of Brazil, which is the most populated region of the country. Surface water samples were taken every 3 months for 1.5 years at four different sites downstream the city of Juiz de Fora. A total of 28 compounds were analyzed on an UHPLC-Orbitrap-MS/MS using a direct injection method. Nine substances were found in at least one water sample, with concentrations ranging from 11 to 4471 ng L-1. The micropollutants found in the river were not detected at the reference site upstream of the city, except for caffeine, which was present at low concentrations in the reference site. Additionally, a nontarget screening of the river samples was applied, which resulted in the identification of 116 chemicals, most of which were pharmaceuticals. Concentrations of most of the micropollutants varied with season and correlated significantly with rainfall events, which caused dilution in the river. The highest observed concentrations were for pharmaceuticals used for treating chronic diseases, such as metformin, which is used to treat diabetes, and were among the most consumed in Juiz de Fora during the study period. Moderate ecotoxicological risks were found for metformin, oxazepam, triclosan, and tramadol. Considering the complex mixture of micropollutants in the environment, more knowledge is needed to elucidate their ecological risk in aquatic ecosystems.

Amazon River carbon dioxide outgassing fuelled by wetlands.

River systems connect the terrestrial biosphere, the atmosphere and the ocean in the global carbon cycle. A recent estimate suggests that up to 3 petagrams of carbon per year could be emitted as carbon dioxide (CO2) from global inland waters, offsetting the carbon uptake by terrestrial ecosystems. It is generally assumed that inland waters emit carbon that has been previously fixed upstream by land plant photosynthesis, then transferred to soils, and subsequently transported downstream in run-off. But at the scale of entire drainage basins, the lateral carbon fluxes carried by small rivers upstream do not account for all of the CO2 emitted from inundated areas downstream. Three-quarters of the world's flooded land consists of temporary wetlands, but the contribution of these productive ecosystems to the inland water carbon budget has been largely overlooked. Here we show that wetlands pump large amounts of atmospheric CO2 into river waters in the floodplains of the central Amazon. Flooded forests and floating vegetation export large amounts of carbon to river waters and the dissolved CO2 can be transported dozens to hundreds of kilometres downstream before being emitted. We estimate that Amazonian wetlands export half of their gross primary production to river waters as dissolved CO2 and organic carbon, compared with only a few per cent of gross primary production exported in upland (not flooded) ecosystems. Moreover, we suggest that wetland carbon export is potentially large enough to account for at least the 0.21 petagrams of carbon emitted per year as CO2 from the central Amazon River and its floodplains. Global carbon budgets should explicitly address temporary or vegetated flooded areas, because these ecosystems combine high aerial primary production with large, fast carbon export, potentially supporting a substantial fraction of CO2 evasion from inland waters.

The transformation of macrophyte-derived organic matter to methane relates to plant water and nutrient contents.

Macrophyte detritus is one of the main sources of organic carbon (OC) in inland waters, and it is potentially available for methane (CH4) production in anoxic bottom waters and sediments. However, the transformation of macrophyte-derived OC into CH4 has not been studied systematically, thus its extent and relationship with macrophyte characteristics remains uncertain. We performed decomposition experiments of macrophyte detritus from 10 different species at anoxic conditions, in presence and absence of a freshwater sediment, in order to relate the extent and rate of CH4 production to the detritus water content, C/N and C/P ratios. A significant fraction of the macrophyte OC was transformed to CH4 (mean = 7.9%; range = 0-15.0%) during the 59-d incubation, and the mean total C loss to CO2 and CH4 was 17.3% (range = 1.3-32.7%). The transformation efficiency of macrophyte OC to CH4 was significantly and positively related to the macrophyte water content, and negatively to its C/N and C/P ratios. The presence of sediment increased the transformation efficiency to CH4 from an average of 4.0% (without sediment) to 11.8%, possibly due to physicochemical conditions favorable for CH4 production (low redox potential, buffered pH) or because sediment particles facilitate biofilm formation. The relationship between macrophyte characteristics and CH4 production can be used by future studies to model CH4 emission in systems colonized by macrophytes. Furthermore, this study highlights that the extent to which macrophyte detritus is mixed with sediment also affects CH4 production.

Environmental Risk of Metal Contamination in Sediments of Tropical Reservoirs.

Reservoir sediment can work as both sink and source for contaminants. Once released into the water column, contaminants can be toxic to biota and humans. We investigate potential ecological risk to benthic organisms by metals contamination in six reservoirs in Southeast Brazil. Results of the bioavailable fraction of copper (Cu), chromium (Cr), cadmium (Cd), lead (Pb), zinc (Zn), and iron (Fe) in sediment samples are presented. Considering Cu, Cd, and Zn concentrations, about 6% of the samples exceeded the threshold effect levels of sediment quality guidelines. The comparison to sediment quality guidelines is conservative because we used a moderate metal extraction. Control of contaminant sources in these reservoirs is key because they are sources of water and food. The mixture toxicity assessment showed an increased incidence of toxicity to aquatic organisms showing that mixture toxicity should be taken into account in sediment assessment criteria.

Large but variable methane production in anoxic freshwater sediment upon addition of allochthonous and autochthonous organic matter.

An important question in the context of climate change is to understand how CH4 production is regulated in anoxic sediments of lakes and reservoirs. The type of organic carbon (OC) present in lakes is a key factor controlling CH4 production at anoxic conditions, but the studies investigating the methanogenic potential of the main OC types are fragmented. We incubated different types of allochthonous OC (alloOC; terrestrial plant leaves) and autochthonous OC (autoOC; phytoplankton and two aquatic plants species) in an anoxic sediment during 130 d. We tested if (1) the supply of fresh alloOC and autoOC to an anoxic refractory sediment would fuel CH4 production and if (2) autoOC would decompose faster than alloOC. The addition of fresh OC greatly increased CH4 production and the δ13C-CH4 partitioning indicated that CH4 originated exclusively from the fresh OC. The large CH4 production in an anoxic sediment fueled by alloOC is a new finding which indicates that all systems with anoxic conditions and high sedimentation rates have the potential to be CH4 emitters. The autoOC decomposed faster than alloOC, but the total CH4 production was not higher for all autoOC types, one aquatic plant species having values as low as the terrestrial leaves, and the other one having values as high as phytoplankton. Our study is the first to report such variability, suggesting that the extent to which C fixed by aquatic plants is emitted as greenhouse gases or buried as OC in sediment could more generally differ between aquatic vegetation types.

Spatially Resolved Measurements of CO2 and CH4 Concentration and Gas-Exchange Velocity Highly Influence Carbon-Emission Estimates of Reservoirs.

The magnitude of diffusive carbon dioxide (CO2) and methane (CH4) emission from man-made reservoirs is uncertain because the spatial variability generally is not well-represented. Here, we examine the spatial variability and its drivers for partial pressure, gas-exchange velocity (k), and diffusive flux of CO2 and CH4 in three tropical reservoirs using spatially resolved measurements of both gas concentrations and k. We observed high spatial variability in CO2 and CH4 concentrations and flux within all three reservoirs, with river inflow areas generally displaying elevated CH4 concentrations. Conversely, areas close to the dam are generally characterized by low concentrations and are therefore not likely to be representative for the whole system. A large share (44-83%) of the within-reservoir variability of gas concentration was explained by dissolved oxygen, pH, chlorophyll, water depth, and within-reservoir location. High spatial variability in k was observed, and kCH4 was persistently higher (on average, 2.5 times more) than kCO2. Not accounting for the within-reservoir variability in concentrations and k may lead to up to 80% underestimation of whole-system diffusive emission of CO2 and CH4. Our findings provide valuable information on how to develop field-sampling strategies to reliably capture the spatial heterogeneity of diffusive carbon fluxes from reservoirs.

Microbial Food-Web Drivers in Tropical Reservoirs.

Element cycling in aquatic systems is driven chiefly by planktonic processes, and the structure of the planktonic food web determines the efficiency of carbon transfer through trophic levels. However, few studies have comprehensively evaluated all planktonic food-web components in tropical regions. The aim of this study was to unravel the top-down controls (metazooplankton community structure), bottom-up controls (resource availability), and hydrologic (water residence time) and physical (temperature) variables that affect different components of the microbial food web (MFW) carbon stock in tropical reservoirs, through structural equation models (SEM). We conducted a field study in four deep Brazilian reservoirs (Balbina, Tucuruí, Três Marias, and Funil) with different trophic states (oligo-, meso-, and eutrophic). We found evidence of a high contribution of the MFW (up to 50% of total planktonic carbon), especially in the less-eutrophic reservoirs (Balbina and Tucuruí). Bottom-up and top-down effects assessed through SEM indicated negative interactions between soluble reactive phosphorus and phototrophic picoplankton (PPP), dissolved inorganic nitrogen, and heterotrophic nanoflagellates (HNF). Copepods positively affected ciliates, and cladocerans positively affected heterotrophic bacteria (HB) and PPP. Higher copepod/cladoceran ratios and an indirect positive effect of copepods on HB might strengthen HB-HNF coupling. We also found low values for the degree of uncoupling (D) and a low HNF/HB ratio compared with literature data (mostly from temperate regions). This study demonstrates the importance of evaluating the whole size spectrum (including microbial compartments) of the different planktonic compartments, in order to capture the complex carbon dynamics of tropical aquatic ecosystems.

Environmental rather than spatial factors structure bacterioplankton communities in shallow lakes along a > 6000 km latitudinal gradient in South America.

Metacommunity studies on lake bacterioplankton indicate the importance of environmental factors in structuring communities. Yet most of these studies cover relatively small spatial scales. We assessed the relative importance of environmental and spatial factors in shaping bacterioplankton communities across a > 6000 km latitudinal range, studying 48 shallow lowland lakes in the tropical, tropicali (isothermal subzone of the tropics) and tundra climate regions of South America using denaturing gradient gel electrophoresis. Bacterioplankton community composition (BCC) differed significantly across regions. Although a large fraction of the variation in BCC remained unexplained, the results supported a consistent significant contribution of local environmental variables and to a lesser extent spatial variables, irrespective of spatial scale. Upon correction for space, mainly biotic environmental factors significantly explained the variation in BCC. The abundance of pelagic cladocerans remained particularly significant, suggesting grazer effects on bacterioplankton communities in the studied lakes. These results confirm that bacterioplankton communities are predominantly structured by environmental factors, even over a large-scale latitudinal gradient (6026 km), and stress the importance of including biotic variables in studies that aim to understand patterns in BCC.

Visualizing aquatic bacteria by light and transmission electron microscopy.

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.

Cross-continental importance of CH4 emissions from dry inland-waters.

Despite substantial advances in quantifying greenhouse gas (GHG) emissions from dry inland waters, existing estimates mainly consist of carbon dioxide (CO2) emissions. However, methane (CH4) may also be relevant due to its higher Global Warming Potential (GWP). We report CH4 emissions from dry inland water sediments to i) provide a cross-continental estimate of such emissions for different types of aquatic systems (i.e., lakes, ponds, reservoirs, and streams) and climate zones (i.e., tropical, continental, and temperate); and ii) determine the environmental factors that control these emissions. CH4 emissions from dry inland waters were consistently higher than emissions observed in adjacent uphill soils, across climate zones and in all aquatic systems except for streams. However, the CH4 contribution (normalized to CO2 equivalents; CO2-eq) to the total GHG emissions of dry inland waters was similar for all types of aquatic systems and varied from 10 to 21%. Although we discuss multiple controlling factors, dry inland water CH4 emissions were most strongly related to sediment organic matter content and moisture. Summing CO2 and CH4 emissions revealed a cross-continental average emission of 9.6 ± 17.4 g CO2-eq m-2 d-1 from dry inland waters. We argue that increasing droughts likely expand the worldwide surface area of atmosphere-exposed aquatic sediments, thereby increasing global dry inland water CH4 emissions. Hence, CH4 cannot be ignored if we want to fully understand the carbon (C) cycle of dry sediments.

Increasing Temperature Counteracts the Negative Effect of UV Radiation on Growth and Photosynthetic Efficiency of Microcystis aeruginosa and Raphidiopsis raciborskii.

High temperature can promote cyanobacterial blooms, whereas ultraviolet radiation (UVR) can potentially depress cyanobacterial growth by damaging their photosynthetic apparatus. Although the damaging effect of UVR has been well documented, reports on the interactive effects of UV radiation exposure and warming on cyanobacteria remain scarce. To better understand the combined effects of temperature and UVR on cyanobacteria, two strains of nuisance species, Microcystis aeruginosa (MIRF) and Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii, CYRF), were grown at 24°C and 28°C and were daily exposed to UVA + UVB (PAR + UVA+UVB) or only UVA (PAR + UVA) radiation. MIRF and CYRF growth rates were most affected by PAR + UVA+UVB treatment and to a lesser extent by the PAR + UVA treatment. Negative UVR effects on growth, Photosystem II (PSII) efficiency and photosynthesis were pronounced at 24°C when compared to that at 28°C. Our results showed a cumulative negative effect on PSII efficiency in MIRF, but not in CYRF. Hence, although higher temperature ameliorates UVR damage, interspecific differences may lead to deviating impacts on different species, and combined elevated temperature and UVR stress could influence species competition.

Virus-bacterium coupling driven by both turbidity and hydrodynamics in an Amazonian floodplain lake.

The importance of viruses in aquatic ecosystem functioning has been widely described. However, few studies have examined tropical aquatic ecosystems. Here, we evaluated for the first time viruses and their relationship with other planktonic communities in an Amazonian freshwater ecosystem. Coupling between viruses and bacteria was studied, focusing both on hydrologic dynamics and anthropogenic forced turbidity in the system (Lake Batata). Samples were taken during four hydrologic seasons at both natural and impacted sites to count virus-like particles (VLP) and bacteria. In parallel, virus-infected bacteria were identified and quantified by transmission electron microscopy (TEM). Viral abundance ranged from 0.5 × 107 ± 0.2 × 107 VLP ml⁻¹ (high-water season, impacted site) to 1.7 × 107 ± 0.4 × 107 VLP ml⁻¹ (low-water season, natural site). These data were strongly correlated with the bacterial abundance (r² = 0.84; P < 0.05), which ranged from 1.0 × 106 ± 0.5 × 106 cells ml⁻¹ (high water, impacted site) to 3.4 × 106 ± 0.7 × 106 cells ml⁻¹ (low water, natural site). Moreover, the viral abundance was weakly correlated with chlorophyll a, suggesting that most viruses were bacteriophages. TEM quantitative analyses revealed that the frequency of visibly infected cells was 20%, with 10 ± 3 phages per cell section. In general, we found a low virus-bacterium ratio (<7). Both the close coupling between the viral and bacterial abundances and the low virus-bacterium ratio suggest that viral abundance tends to be driven by the reduction of hosts for viral infection. Our results demonstrate that viruses are controlled by biological substrates, whereas in addition to grazing, bacteria are regulated by physical processes caused by turbidity, which affect underwater light distribution and dissolved organic carbon availability.

Sediment drying-rewetting cycles enhance greenhouse gas emissions, nutrient and trace element release, and promote water cytogenotoxicity.

Increased periods of prolonged droughts followed by severe precipitation events are expected throughout South America due to climate change. Freshwater sediments are especially sensitive to these changing climate conditions. The increased oscillation of water levels in aquatic ecosystems causes enhanced cycles of sediment drying and rewetting. Here we experimentally evaluate the effects of induced drought followed by a rewetting event on the release of carbon dioxide (CO2), methane (CH4), nutrients (nitrogen and phosphorus), and trace elements (iron, manganese, and zinc) from the sediment of a tropical reservoir in southeastern Brazil. Furthermore, we used bulb onions (Allium cepa) to assess the potential cytogenotoxicity of the water overlying sediments after rewetting. We found peaks in CO2 and CH4 emissions when sediments first transitioned from wet to dry, with fluxes declining as sediments dried out. CO2 emissions peaked again upon rewetting, whereas CH4 emissions remained unaltered. Our experiment also revealed average increases by up to a factor of ~5000 in the release rates of nutrients and trace elements in water overlying sediments after rewetting. These increased release rates of potentially toxic compounds likely explain the lower replication of Allium cepa cells (up to 22% reduction) exposed to water overlying sediments after rewetting. Our findings suggest that increased events of drought followed by rewetting may lead to a range of changes in freshwater ecosystems, including nutrient enrichment, increased toxicity following resuspension of contaminants, and higher emission of greenhouse gases to the atmosphere.

A global trend of caffeine consumption over time and related-environmental impacts.

Caffeine is one of the most consumed substances, and it has been largely detected in aquatic ecosystems. We investigated the trends in caffeine consumption over three decades and its relationships with gross domestic product (GDP) and human development index (HDI) to understand global patterns and to identify potential hotspots of contamination. The total caffeine consumption is increasing mainly due to population growth. Moreover, caffeine consumption per capita is also increasing in some countries, such as Brazil, Italy, and Ethiopia. A high positive correlation between caffeine consumption per capita with HDI and GDP was found for coffee-importing countries in Europe, while a high negative correlation was found for coffee-exporting countries in Africa. The literature review showed that the highest caffeine concentrations coincide with countries that present an increasing caffeine consumption per capita. Also, approximately 35% of the caffeine concentrations reported in the literature were above the predicted no-effect concentration in the environment and, again, overlaps with countries with increasing per capita consumption. Despite the high degradation rate, caffeine consumption tends to increase in a near future, which may also increase the overall amount of caffeine that comes into the environment, possibly exceeding the thresholds of several species described as tolerant to the current environmental concentrations. Therefore, it is essential to prevent caffeine from reaching aquatic ecosystems, implementing sewage treatment systems, and improving their efficiency.

Investigation of medicines consumption and disposal in Brazil: A study case in a developing country.

The incorrect disposal of medicines can be harmful to the environment. Here, we aim to understand the consumption and disposal of medicines in Brazil using online forms. 64% of the respondents have the habit to self-medicate. 66% of respondents dispose the disused or expired medicines in the garbage. 71.9% of respondents never receive any information about correct disposal of medicines. 95.2% of respondents believe that residues of medicines can be harmful to the environment. Environmental education can provide information to the population and help to mitigate pharmaceuticals pollution.

Limnological effects of a large Amazonian run-of-river dam on the main river and drowned tributary valleys.

Run-of-river dams are often considered to have lower environmental impacts than storage dams due to their smaller reservoirs and low potential for flow alteration. However, this has been questioned for projects recently built on large rivers around the world. Two of the world's largest run-of-river dams-Santo Antônio and Jirau-were recently constructed on the Madeira River, a major tributary to the Amazon River in Brazil. Here we evaluate the effects of the creation of the Santo Antônio dam on the water chemistry and thermal structure of the Madeira River mainstem and back-flooded valleys of tributaries within the reservoir inundated area. In contrast to the mainstem river, some back-flooded tributaries periodically developed thermal stratification, which is associated with higher water residence times. Additionally, biochemical oxygen demand, partial pressure of CO2, and organic carbon all increased in the tributary valleys inundated by the reservoir, possibly due to increased input of allochthonous organic matter and its subsequent mineralization upon back-flooding-a common feature of newly flooded impoundments. The mainstem did not show detectable dam-related changes in water chemistry and thermal structure. Although the majority of the reservoir area maintained riverine conditions, the lateral valleys formed upon back-flooding-corresponding to ~30% of the Santo Antônio reservoir area-developed lake-like conditions akin to a typical reservoir of a storage dam.

Far-reaching cytogenotoxic effects of mine waste from the Fundão dam disaster in Brazil.

On November 2015, one of Brazil's most important watersheds was impacted by the mine waste from Fundão dam collapse in Mariana. The mine waste traveled over 600 km along the Doce River before reaching the sea, causing severe devastation along its way. Here we assessed trace element concentrations and cytogenotoxic effects of the released mine waste. Water samples were collected along the Doce River ten days after the disaster in two impacted sites and one non-impacted site. Sampling points were located hundreds of kilometers downstream of the collapsed dam. Water samples were used for trace element quantification and to run an experiment using Allium cepa to test cytogenotoxicity. We found extremely high concentrations of particulate Fe, Al, and Mn in the impacted sites. We observed cytogenotoxic effects such as alterations in mitotic and phase indexes, and enhanced frequency of chromosomal aberrations. Our results indicate interferences in the cell cycle in impacted sites located hundreds of kilometers downstream of the disaster. The environmental impacts of the dam collapse may not only be far-reaching but also very likely long-lasting, because the mine waste may persist in the Doce River sediment for decades.

Histological approaches for high-quality imaging of zooplanktonic organisms.

The investigation of the internal organization of zooplankton communities provides important information on the plankton biology with special interest for the study of ecological processes. Zooplanktoners can play a structural function as indicators for ecosystem health or stress, but their study using histological techniques is still limited. Here we report that the internal structure of zooplanktonic organisms can be facilely observed by a histological approach that combines optimal fixation and processing with a plastic resin (glycol methacrylate) embedding, resulting in increased tissue resolution. Using copepods, organisms that can dominate zooplankton assemblages, as models, collected from a tropical ecosystem (Paraibuna river, Brazil), we showed fine histological details of their muscular, nervous and digestive systems, structure of appendages and cell features. Critical advantages of this approach are that it permits optimal preservation and adequate handling of the organisms (embedded in agar after fixation) for further histological processing and investigation. This is important because it prevents both mechanically induced artifacts and loss of these diminutive organisms during the different steps of processing. Moreover, embedding in plastic resin showed a superior imaging of copepod internal structures compared to paraffin embedding. The use of glycol methacrylate is advantageous over paraffin/paraplast embedding by avoiding heat damage, tissue retraction and allowing faster embedding procedure and better tissue resolution. The value of histological approaches in enabling high-quality imaging of the internal structure of copepods is particularly important because these organisms can be used as indicators of environmental changes.