Changes in cell-type composition in digestive gland of slugs and its influence in biomarkers following transplantation between a relatively unpolluted and a chronically metal-polluted site

Changes in cell-type composition (CCTC) is a general phenomenon that takes place in the digestive gland epithelium of stressed molluscs. The aim of the present work was to determine whether CCTC is a reversible process in the digestive gland of sentinel slugs chronically exposed to metal pollution and how CCTC affects metal accumulation parameters and different cell and tissue biomarkers of exposure and effect. Slugs (Arion ater) from an abandoned zinc mine were transferred to a relatively unpolluted site and the other way around for 3, 10 and 28 d. The volume density of black silver deposits (Vv_BSD) after autometallography, and metallothionein (MT) levels were used as biomarkers of exposure to metals and CCTC and lysosomal responses were selected as effect biomarkers. Results indicated that slugs were sensitive to recent metal pollution; however, slugs chronically exposed to metals presented some characteristic features and were less responsive to pollution cessation without signs of CCTC reversal.     

Localization of Hg and Pb in the palps,the digestive gland and the gills in Mytilus galloprovincialis (L.) using autometallography and X-ray microanalysis

The intracellular localization of heavy metals using autometallography (AMG) and X-ray microanalysis was studied in the palps, the digestive gland and the gills of mussels Mytilus galloprovincialis, after an experimental exposure to 0.1 mg l(-1) of Hg and 0.1 mg l(-1) of Pb, for 30 and 60 days. In the examined tissues, autometallographical black silver deposits (BSD) were localized mainly in the residual bodies and heterolysosomes of the digestive cells, as well as in the dense bodies of the epithelial cells. Metal deposition after Hg exposure was much more abundant compared to Pb exposure. Using X-ray microanalysis, Hg was traced on the BSD in all examined tissues, while Pb was not traced in these deposits. The results are discussed in comparison to previous results on long-term exposure to the same metals; in addition, the palps are introduced as a new metal storing organ and, finally, the use of X-ray microanalysis under a scanning electron microscope in order to enhance the specificity of AMG is suggested.     

Comparative study of the accumulation and detoxification of Cu (essential metal) and Hg (nonessential metal) in the digestive gland and gills of mussels Mytilus galloprovincialis, using analytical and histochemical techniques

The aim of the present study is the comparative examination of accumulation and detoxification of Cu and Hg in digestive gland and gills of mussels Mytilus galloprovincialis, using atomic absorption spectrophotometry and autometallography. Mussels were exposed to 0.08 mg L⁻¹ Cu, 0.08 mg L⁻¹ Hg, as well as to a mixture of 0.08 mg L⁻¹ Hg and 0.08 mg L⁻¹ Cu for 11 d. After the experimental exposure, animals were kept under laboratory conditions for a detoxification period of 7 d. An antagonistic effect of Cu against to Hg accumulation was noted in the digestive gland of mussels after the experimental exposure, as well as after the detoxification period, supporting the protective role of Cu against to Hg toxicity in this tissue. Digestive gland was suggested as a main organ for Hg accumulation and gills as a target position for Cu accumulation. Additionally, lower time was evaluated for Hg detoxification in the digestive gland and gills of mussels, in relation to those addressed for Cu detoxification in the same tissues. The evaluation of black silver deposits (BSD) extent performed in digestive gland and gills was suggested as a less sensitive approach, in relation to atomic absorption spectrophotometry (AAS), to indentify the concentration of heavy metals in tissues of mussels. The toxic effects of Hg, Cu and a mixture of them on lysosomal system of the digestive cells are also discussed.     

Lysosomal enlargement and lysosomal membrane destabilisation in mussel digestive cells measured by an integrative index

Lysosomal responses (enlargement and membrane destabilisation) in mussel digestive cells are well-known environmental stress biomarkers in pollution effects monitoring in marine ecosystems. Presently, in laboratory and field studies, both responses were measured separately (in terms of lysosomal volume density - Vv - and labilisation period -LP) and combined (lysosomal response index - LRI) in order to contribute to their understanding and to develop an index useful for decisions makers. LRI integrates Vv and LP, which are not necessarily dependent lysosomal responses. It is unbiased and more sensitive than Vv and LP alone and diminishes background due to confounding factors. LRI provides a simple numerical index (consensus reference = 0; critical threshold = 1) directly related to the pollution impact degree. Moreover, LRI can be represented in a way that allows the interpretation of lysosomal responses, which is useful for environmental scientists.     

The role of algae (Isochrysis galbana) enrichment on the bioaccumulation of benzo[a]pyrene and its effects on the blue mussel Mytilus edulis

The role of algal concentration in the transfer of organic contaminants in a food chain has been studied using the ubiquitous model polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) as the contaminant, Isochrysis galbana as the phytoplankton food source, and the common mussel (Mytilus edulis) as the primary consumer. The effect of algal concentration on BaP uptake by M. edulis was determined by feeding M. edulis daily with I. galbana which had previously been kept in the presence of BaP for 24 h. Four combinations of concentrations of algae and BaP were used to give final exposure concentrations of 30,000 or 150,000 algal cells ml(-1) in combination with either 2 or 50 microg BaP l(-1). BaP concentrations were determined fluorometrically in rest tissues (excluding digestive glands) and digestive gland microsomal fractions of M. edulis after 1, 7 and 15 days exposure, and also in isolated algae. Potentially toxic effects of BaP on M. edulis were examined in terms of blood cell lysosomal membrane damage (neutral red dye retention assay) and induction of digestive gland microsomal mixed-function oxygenase (MFO) parameters [BaP hydroxylase (BPH) and NADPH-cytochrome c (P450) reductase activities]. BaP bioaccumulation in rest tissues (and to a lesser extent in digestive gland microsomes) of M. edulis increased with both increasing BaP and algal exposure concentrations, and over time, producing maximal bioconcentration factors in rest tissues after 15 days exposure to 150,000 algal cells ml(-1) and 50 microg BaP l(-1) of 250,000. The five-fold higher concentration of algae increased BaP bioaccumulation by a factor of approximately 2 for 50 microg BaP l(-1) at day 15. Blood cell neutral red dye retention time decreased linearly with increasing log(10) tissue BaP body burden, indicating an increased biological impact on M. edulis with increasing BaP exposure possibly due to a direct effect of BaP on blood cell lysosomal membrane integrity. An increase was seen in NADPH-cytochrome c reductase activity, and indicated in BPH activity, with 1 but not 7 or 15 days exposure to BaP, indicating a transient response of the digestive gland microsomal MFO system to BaP exposure.     

Assessment of the mechanisms of detoxification of chemical compounds and antioxidant enzymes in the digestive gland of mussels, Mytilus galloprovincialis, from Mediterranean coastal sites

In this study the effects of the main marine pollutants (metals, PAHs, PCBs and DDTs) were assessed in native mussels from the Mediterranean coast of Spain. For this purpose several biomarkers such as benzo[a]pyrene hydroxylase (BPH), DT-diaphorase (DTD), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPs), glutathione reductase (GR), metallothionein (MT) and lipid peroxidation (LPO) were measured in the digestive gland. Results showed increased LPO levels in mussels which accumulated high loads of organic compounds and arsenic in their tissues. BPH levels correlated to the concentrations of organic compounds in mussel tissues, though the range of BPH response was low in relation to the high gradient of accumulation of organic pollutants. Increased BPH levels, concomitant to low DTD and GST activities, were detected in mussels which presented high levels of organic pollutants in their tissues. This suggests that signs of LPO present in these organisms are related to the imbalance between phase I and phase II biotransformation processes. Furthermore, the increased levels of MT and CAT detected in mussels which showed high levels of Cd in their tissues appear to reflect a coordinated response which protects against the toxicity of this metal. The application of these biomarkers in environmental assessment is discussed.     

Proteomic changes in <Emphasis Type="Italic">Corbicula fluminea</Emphasis> exposed to wastewater from a psychiatric hospital

The increase use of pharmaceutical compounds in veterinary practice and human population results in the ubiquitous presence of these compounds in aquatic ecosystems. Because pharmaceuticals are highly bioactive, there is concern about their toxicological effects in aquatic organisms. Therefore, the aim of this study was to assess the effects of an effluent from a psychiatric hospital (containing a complex mixture of 25 pharmaceutical compounds from eleven therapeutic classes) on the freshwater clam Corbicula fluminea using a proteomic approach. The exposure of C. fluminea to this complex effluent containing anxiolytics, analgesics, lipid regulators, beta blockers, antidepressants, antiepileptics, antihistamines, antihypertensives, antiplatelets and antiarrhythmics induced protein changes after 1 day of exposure in clam gills and digestive gland more evident in the digestive gland. These changes included increase in the abundance of proteins associated with structural (actin and tubulin), cellular functions (calreticulin, proliferating cell nuclear antigen (PCNA), T complex protein 1 (TCP1)) and metabolism (aldehyde dehydrogenase (ALDH), alcohol dehydrogenase, 6 phosphogluconate dehydrogenase). Results from this study indicate that calreticulin, PCNA, ALDH and alcohol dehydrogenase in the digestive gland and T complex protein 1 (TCP1)) and 6 phosphogluconate dehydrogenase in the gills represent useful biomarkers for the ecotoxicological characterization of psychiatric hospital effluents in this species.     

Assessment of biological effects of environmental pollution along the NW Mediterranean Sea using mussels as sentinel organisms

With the aim of assessing the biological effects of pollution along three gradients of pollution in the NW Mediterranean Sea, a biomonitoring survey was implemented using a battery of biomarkers (lysosomal membrane stability, lysosomal structural changes, metallothionein (MT) induction and peroxisome proliferation) in mussels over a period of two years as part of the EU-funded BEEP project. Mussels from the most impacted zones (Fos, Genova and Barcelona harbours) showed enlarged lysosomes accompanied by reduced labilisation period of lysosomal membranes, indicating disturbed health. MT levels did not reveal significant differences between stations and were significantly correlated with gonad index, suggesting that they were influenced by gamete development. Peroxisomal acyl-CoA oxidase (AOX) activity was significantly inhibited in polluted stations possibly due to interactions among mixtures of pollutants. In conclusion, the application of a battery of effect and exposure biomarkers provided relevant data for the assessment of biological effects of environmental pollution along the NW Mediterranean Sea.     

可吸附有机卤化物的深度处理实验研究

可吸附有机卤化物（AOX）是人为污染的重要标志之一,北京高碑店污水处理厂二级出水中约90％的AOX为可吸附有机氧化物（AOCl),研究了自氧氧化,粒状活性炭吸附,粉末活性炭吸附3种深度处理工艺对二级出水中AOX的去除作用,臭氧的氧化反应最多可去除约38％的AOX,粒状活性炭床可运行3200床体积,吸附容量为0.14mgAOX/g GH-16型活性炭,投加木质粉末活性炭200mg/L及25mg/L的聚合氯化铝,能去除24．7％的AOX。     

Cytotoxicity assessment of four pharmaceutical compounds on the zebra mussel (Dreissena polymorpha) haemocytes, gill and digestive gland primary cell cultures

Pharmaceutical compounds are considered the new environmental pollutants but at present few studies have evaluated their ecotoxicity on aquatic invertebrates. This study was aimed to investigate the in vitro cytotoxicity of four common drugs, namely atenolol (ATL), carbamazepine (CBZ), diclofenac (DCF) and gemfibrozil (GEM), on three different cell typologies from the zebra mussel (Dreissena polymorpha): haemocytes, gill and digestive gland cells. Results obtained by the Trypan blue exclusion test revealed that exposure to increasing concentrations (0.001; 0.01; 0.1; 1 and 10 mg L⁻¹) of CBZ, DCF and GEM were able to significantly decrease the viability of each cell type, while the MTT (3(4,5-dimethyl-2thiazholyl)-2,5-diphenyl-2H-tetrazolium bromide) reduction assay highlighted only a slight reduction of mitochondrial activity of gill and digestive gland cells. Overall, DCF was the most cytotoxic drug for zebra mussel cells, followed by GEM, CBZ, while ATL has not a noteworthy toxic potential. Our preliminary results lay the groundwork for further in vitro evaluations, which will allow a better definition of the potential toxicity of these drugs.     

Assessment of typical pollutants in waterborne by combining active biomonitoring and integrated biomarkers response

Organic pollutants, heavy metals and pharmaceuticals are continuously dispersed into the environment and have become a relevant environmental emerging concern. In this study, a situ assay to assess ecotoxicity of mixed pollutants was carried out in three typical sites with different priority contaminations in Guangzhou, China. Chemical analysis of organic pollutants, metals and quinolones in three exposure sites were determined by GC-ECD/MS, ICP-AES and HPLC, as well as, a combination of biomarkers including: ethoxyresorufin O-deethylase (EROD); aminopyrine N-demethylase (APND); erythromycin N-demethylase (ERND); glutathione S-transferase (GST); malondialdehyde (MDA); CYP1A; and P-glycoprotein (P-gp) mRNA expressions were evaluated in Mugilogobius abei. Results of chemical analysis in sediment samples revealed that the dominant chemicals were organic pollutants and heavy metals in Huadi River while quinolones in the pond. Bioassays indicated that differences among sites were in relation to some specific biomarkers. EROD and GST activities significantly increased after 72 h in situ exposure, but no difference was observed among the exposure sites. APND, ERND and MDA exhibited dissimilar change patterns for different priority pollutants. CYP1A and P-gp mRNA expressions were significantly induced at all exposure sites, whilst P-gp activity was typical for S2 with the highest levels of quinolones. The molecular biomarkers seemed to be more susceptible than enzyme activities. These assays confirmed the usefulness of applying a large array of various combined biomarkers at different levels, in assessing the toxic effects of mixed pollutants in a natural aquatic environment.     

Effects of atmospheric exposure to naphthalene on xenobiotic-metabolising enzymes in the snail Helix aspersa

Polycyclic aromatic hydrocarbons are xenobiotics whose elevated toxicity for living organisms requires to efficiently monitor air pollution, either by evaluating their levels in the environment, or by assessing their biological impacts on sentinel organisms. We investigated the effects of naphthalene exposure on some xenobiotic-metabolising enzyme activities in three organs of Helix aspersa. Particular activities depending on cytochrome P450 (ethoxyresorufin O-deethylase, EROD; ethoxycoumarin O-deethylase, ECOD; pentoxyresorufin O-dealkylase, PROD) and associated with glutathione (glutathione S-transferase, GST; glutathione peroxidase, GPX; glutathione reductase, GR) were assessed. In control animals, the P450-dependent specific activities were distributed according to the range kidney > digestive gland > mantle cavity forming tissues (MCFT). Neither ECOD nor PROD activities could be detected in MCFT. In the two other organs, the major phase I activities were due to ECOD, the level of PROD being very low or null. The glutathione-associated activities showed comparable levels in the three organs, except GPX activity that was higher in the digestive gland. Naphthalene (NAP) exposure did not affect any activity in MCFT, but it significantly decreased EROD and ECOD activities in the kidney as opposed to their increase in the digestive gland, whereas PROD activities were not influenced by the treatment. Glutathione-dependent activities were not significantly affected by NAP exposure, except for GPX which activity diminished in the digestive gland. This study demonstrates that complex detoxification pathways should exist in Helix aspersa as in mammals and that they could be used as potential biomarkers of NAP exposure.     

Lysosomal and lipid alterations in the digestive gland of mussels, Mytilus galloprovincialis (L.) as biomarkers of environmental stress

Lysosomal and lipid alterations in the digestive gland cells of the common mussel Mytilus galloprovincialis collected from six stations along Thermaikos Gulf (Northern Greece) were monitored through a 1-year period. The ultrastructural observations of the digestive cells of sampled mussels indicated a higher incidence of "granular" or "dark" residual bodies in the relatively more polluted stations compared to less polluted ones. Alterations in the lysosomal system of digestive cells were assessed: (1) using the lysosomal membrane stability test (2) quantifying lysosomal structural alterations of the residual bodies and the heterolysosomes of the digestive cells at the ultrastructural level, and (3) determining the elemental composition of lysosomes by means of X-ray microanalysis on epoxy resin sections. Evaluation of neutral lipids was realized using image analysis on histochemical resin sections. Mussels from the most heavily polluted stations exhibited reduced lysosomal membrane stability, higher volume density and diameter of heterolysosomes and residual bodies, and, in some cases, relatively higher lysosomal heavy metal content compared to mussels from less polluted stations. The results showed that the lysosomal membrane stability and the volume density of the residual bodies are more sensitive to general stress compared to the other biomarkers used, and could be used successfully as early warning tools in biomonitoring programs.     

Impaired glutathione redox status is associated with decreased survival in two organophosphate-poisoned marine bivalves

Biomonitoring organophosphate (OP) exposure in marine environments is generally achieved by the measurement of acetylcholinesterase activity in bivalves like mussels. However, there is evidence that indicates that oxidative stress may be implied in OP toxicity. The aim of this study was to evaluate the relationship between survival from the OP insecticide fenitrothion and glutathione levels in marine bivalves. Mussels (Mytilus galloprovincialis Lam.) and scallops (Flexopecten flexuosus Poli) were exposed, in a time to death test, to their LC85 of fenitrothion for 96 h. OP-poisoned mussels showed reduced (GSH) and oxidised (GSSG) glutathione depletion in the digestive gland, muscle and gills. Pectinid spats exposed to this insecticide presented GSH depletion in the digestive gland and mantle, and a reduction of the GSH/GSSG ratio in gills and mantle. Although survival curves were significantly different and mussels withstood twice as much fenitrothion as pectinid spats, muscular GSH/GSSG ratio was highly related to mortality in both species. We suggest that an impairment in the glutathione redox status could result in an induction of the cell death, either by apoptosis or necrosis, leading ultimately to the death of the organism. We conclude that whereas glutathione depletion can be used as a biomarker of exposure, the muscular GSH/GSSG ratio might be used as a biochemical marker of effect and individual susceptibility to mortality of marine bivalves exposed to fenitrothion or other pollutants that induce oxidative stress.     

Effects of permethrin exposure on antioxidant enzymes and protein status in Mediterranean clams Ruditapes decussatus

The effects of permethrin (PER) on a panel of antoxidant enzymes; superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and indices of protein oxidation status (carbonylation and free thiols) were determined in digestive gland and gills of the clam Ruditapes decussatus. Animals were exposed to 100 ppb PER for 2 days. These enzyme activities increased significantly in digestive gland (p?<?0.05) after PER treatment and oxidative modification of proteins was detected in both gill and digestive gland extracts using redox proteomics. PER exposure significantly reduced the amount of protein free thiol groups in digestive gland rather than in gill, when compared to controls. Conversely, digestive gland showed significantly higher levels of carbonylated proteins than gill after PER exposure. Some proteins were successfully identified by mass spectrometry of tryptic peptides. Our data suggest that digestive gland of R. decussatus can be used as a model tissue for investigating environmental risk of PER contamination.     

Ubiquitination and carbonylation of proteins in the clam Ruditapes decussatus, exposed to nonylphenol using redox proteomics

Ubiquitination and carbonylation of proteins were investigated in the gill and digestive gland of Ruditapes decussatus exposed to NP (nonylphenol) (100 μgL(-1)) using redox proteomics. After 21 d of exposure, clams were dissected and cytosolic proteins of both tissues separated by 2DE SDS-PAGE. Protein expression profiles were tissue-dependent and differently affected by NP exposure. Ubiquitination and carbonylation were also tissue-specific. NP exposure induced significantly more ubiquitinated proteins in gills than in digestive glands, compared to controls. Digestive gland showed a significant higher number of carbonylated proteins than gills after NP exposure. Protein ubiquitination and carbonylation are therefore independent processes. Results showed that NP exposure generated ROS in gill and digestive gland of R. decussatus that significantly altered the proteome. Results also highlighted the advantage of using redox proteomics in the assessment of protein ubiquitination and carbonylation, which may be markers of oxidative stress in R. decussatus.     

AHH activity, tissue dose and DNA damage in different tissues of the scallop Chlamys farreri exposed to benzo[a]pyrene

A collaborative study was performed on scallops (Chlamys farreri) exposed to 0.5, 3 and 10 microg/L benzo[a]pyrene (B[a]P) for 20 days. The levels of aryl hydrocarbon hydroxylase (AHH) activity, B[a]P accumulation and DNA strand break were assayed in the gill and digestive gland. Results showed that AHH activity and B[a]P accumulation were significantly related to B[a]P dose. AHH activity was induced and then became stable gradually. B[a]P accumulation increased first and showed an incoming plateau. DNA strand break levels in the 0.5 and 3 microg/L B[a]P groups remained high and significantly different from control values until day 6, followed by a reduction in the gill and no recovery in the digestive gland. The 10 microg/L B[a]P groups remained significantly lower than control until the end. These results suggested that the application of comprehensive indices may give information on the actual exposure of organisms to pollutants, and also information on toxic effects.     

The effects of endosulfan on the great ramshorn snail Planorbarius corneus (Gastropoda, Pulmonata): a histopathological study

In this study the great ramshorn snail (Planorbarius corneus), one of the most abundant gastropod of Turkish limnic systems, was investigated to determine the histopathological effects of endosulfan on the digestive gland, foot and mantle under laboratory conditions. Samples were collected from small artificial pools in Karaot at Gelendost-Isparta (southwest of Turkey), where agricultural activities are widespread. The snails were exposed to two sublethal concentrations of endosulfan (0.4 and 0.8 mg/l) for periods of 10, 20 and 30 days. Fifteen snails were kept in 2.5 l glass jars containing dechlorinated tap water and exposed under semi-static test (daily exchange of test water). All the testing was carried out on adult specimens, and snails were maintained on a photothermal period with 16 light hours at 22 +/- 2 degrees C. The histopathological examinations revealed the following changes: amoebocytes infiltration, dilatation in hemolymphatic spaces between the tubules, degeneration of cells, abnormal lumen, necrosis of cells and atrophy in the connective tissue of digestive gland; desquamation of the epithelium cells, changes in the number of mucocytes and protein gland cells, lipid vacuolus and atrophy of the columnar muscle fibers of the foot and mantle tissues. Pycnotic state of cells was also seen in the mantle tissues. Endosulfan caused significant histopathological alterations in the digestive gland, foot and mantle tissues of the snail, irrespective of concentrations of the pesticide and its exposure periods. The results are discussed, particularly in comparison to those of other aquatic organisms.     

New evidences for old biomarkers: effects of several xenobiotics on EROD and AChE activities in Zebra mussel (Dreissena polymorpha)

The biomarker approach is widely used both in vertebrates and invertebrates for environmental biomonitoring, because it can supply an integrated response for multi-xenobiotics contamination. However, the use of biomarkers requires the identification of every possible variation that can influence the biochemical response, because ecosystems are generally subject to a mixture of pollutants, which can create additive, opposite or competitive effects. In recent years, there has been considerable interest in the use of biomarkers within marine bivalves, while very few data are available for freshwater molluscs. The aim of this research was to investigate changes on EROD and AChE activities in the freshwater bivalve Zebra mussel (Dreissena polymorpha) exposed to different pollutants (Arochlor 1260, CB 153 and 126, pp'DDT, chlorpyrifos, carbaryl) at laboratory conditions, in order to standardize the analytical procedures and to highlight eventual interferences on enzyme activities. Chemical concentrations in the mussel soft tissues were analyzed by GC/MS-MS. Main results showed a significant induction of EROD activity when mussels were exposed to 100 ng/l of PCB mixture of Arochlor 1260 and dioxin-like CB 126, but this congener showed also a clear competitive inhibition after 48 h of exposure. Surprisingly, pp'DDT determined a significant decrease of basal EROD activity after only 24 h of exposure, even if it was not possible to discriminate between the effect of the parent compound and that of its metabolites (DDD, DDE). We also found an interaction between the organophosphate insecticide chlorpyrifos, which does not directly decrease the AChE activity, and terbutilazine. This herbicide increased the biotransformation of the organophosphate compound to its oxidized metabolite (oxon), a much stronger AChE inhibitor. The possible use of the oxime Pyridine-2-Aldoxime Methochloride (2-PAM) to bring back the catalytic activity to basal levels was also demonstrated.     

Impact of weathered multi-walled carbon nanotubes on the epithelial cells of the intestinal tract in the freshwater grazers Lymnaea stagnalis and Rhithrogena semicolorata

Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis (L. stagnalis) and the mayfly Rhithrogena semicolorata (R. semicolorata). In L. stagnalis, bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata, irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs (p?<?0.01).