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The current study investigates the severe effects of commonly employed chemicals, such as phenol, on the freshwater bottom-dwelling annelids of Tubifex tubifex. In an acute toxicity test, phenol's 96-hour LC50 value against Tubifex tubifex was identified to be 221.552 mg/L. Using the GUTS simulation, which places the GUTS-SD model on top of the GUTS-IT model, it was possible to confirm that the test organism would survive an acute exposure to phenol overall. After 14 days of treatment with 10 % and 20 % of the phenol's 96-hour LC50 values, long-term bioassays revealed changes in protein levels and in oxidative stress enzyme levels. Total protein concentration dropped during the bioassay, but levels of antioxidant enzymes (CAT, GST, SOD, and MDA) increased. The Pearson correlation matrix and the Integrated Biomarker Response (IBR) index were used for examining the relationship between biomarkers, toxicants, and phenol-induced stress. The results show that exposure to phenol is detrimental to the survival and general health of Tubifex tubifex.

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We investigated the potential ecological risks and harm to aquatic organisms posed by anionic surfactants such as α-olefin sulfonate (AOS), which are commonly found in industrial and consumer products, including detergents. This study assessed acute (96-h) and subchronic (14-day) responses using antioxidant activity, protein levels, and histopathological changes in Tubifex tubifex exposed to different AOS concentrations (10% of the LC50, 20% of the LC50, and a control). Molecular docking was used to investigate the potential interactions between the key stress biomarker enzymes (superoxide dismutase, catalase, and cytochrome c oxidase) of Tubifex tubifex. Acute AOS exposure showed a concentration-dependent decrease in survival, and the general unified threshold (GUTS) model revealed that survivorship is linked to individual response patterns rather than random (stochastic) fluctuations. The GUTS model also revealed dose-dependent toxicity patterns in Tubifex tubifex exposed to α-olefin sulfonate (AOS), with adaptive mechanisms at lower concentrations but significant increases in mortality beyond a certain threshold, emphasizing the role of the AOS concentration in shaping its toxicological impact. Exposure to AOS disrupted antioxidant activity, inducing oxidative stress, with GST and GPx showing positive associations with surfactant concentration and increased lipid peroxidation (elevated MDA levels); moreover, AOS exposure decreased protein concentration, signifying disturbances in vital cellular processes. Histopathological examinations revealed various tissue-level alterations, including cellular vacuolation, cytoplasmic swelling, inflammation, necrosis, and apoptosis. Molecular docking analysis demonstrated interactions between AOS and enzymes (-catalase, superoxide dismutase, and cytochrome c oxidase) in Tubifex tubifex, including hydrophobic and hydrogen bond interactions, with the potential to disrupt enzyme structures and activities, leading to cellular process disruptions, oxidative stress, and tissue damage. According to the species sensitivity distribution (SSD), the difference in toxicity between Tilapia melanopleura (higher sensitivity) and Daphnia magna (low sensitivity) to AOS suggests distinct toxicokinetic and toxicodynamic mechanisms attributable to more complex physiology in Tilapia and efficient detoxification in Daphnia due to its smaller size.

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This study examined the multifaceted impacts of fluorene exposure on Tubifex tubifex, encompassing acute (survival analysis and behavioral responses) and subchronic exposure regimens (antioxidant enzyme response and histopathology), molecular docking studies, and generalized read-across analysis. Survival analysis revealed concentration-dependent increases in toxicity over varying time intervals, with LC50 values decreasing from 30.072 mg/L at 24 h to 12.365 mg/L at 96 h, emphasizing the time-sensitive and concentration-responsive nature of the stressor. Behavioral responses were both concentration- and duration-dependent. While Erratic Movement and Clumping Tendency exhibited earlier responses (within 24 h) at lower concentrations, the wrinkling effect and mucus secretion) exhibited delayed onset, suggesting intricate regulatory mechanisms underlying adaptability to environmental challenges; moreover, the wrinkling effect was consistently induced at higher concentrations, indicating greater sensitivity to the toxic effects of fluorene. With sublethal environmentally relevant concentrations-1.24 mg/l and 2.47 mg/L i.e., 10% and 20% 96 h, respectively-the antioxidant enzyme response (i.e., upregulation of SOD, CAT, and GST) with increasing fluorene concentration, revealing a nonlinear, hormetic response, suggested adaptive protection at lower doses but inhibition at higher concentrations. Histopathological examination indicated that higher fluorene concentrations caused cellular proliferation, inflammation, and severe tissue damage in the digestive tract and body wall. Molecular docking studies demonstrated robust interactions between fluorene and major stress biomarker enzymes, disrupting their functions and inducing oxidative stress. Interactions with cytochrome c oxidase suggested interference with cellular energy production. Generalized Read-Across (GenRA) analysis unveiled shared toxicity mechanisms among fluorene and its analogs, involving the formation of reactive epoxides and the influence of cytochrome P450 enzymes. The diverse functional groups of these analogs, particularly chlorine-containing compounds, were implicated in toxicity through lipid peroxidation and membrane damage. Adverse outcome pathways and broader consequences for aquatic ecosystem health are discussed.

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The improvement effect of Tubifex tubifex on the pollutant removal efficiencies (REs) of vertical flow constructed wetlands (VF-CWs) treating wastewater with various C/N ratios was explored. The experiment was conducted in pilot-scale saturated VF-CWs, being added different densities of T. tubifex and fed synthetic wastewater with successive C/N ratios of 0.5, 1.5, 3.0 and 6.0. The results suggest that T. tubifex addition and the influent C/N ratio had an interactive effect, i.e., T. tubifex addition improved NOx--N, NH4+-N, TN and COD REs by 36.7%, 56.5%, 22.6%, and 10.0%, respectively, under low C/N ratios, while high C/N ratios inhibited this improvement. Low-density T. tubifex addition significantly increased substrate dissolved oxygen (DO) by retarding excessive soil organic matter (OM) accumulation. With T. tubifex addition, an improvement in bacterial diversity, the relative abundance of N-cycle and fermentative bacteria, and N-cycle functional genes was only observed in substrates under low C/N ratios. T. tubifex can improve the purification function of saturated VF-CWs, but this strategy strongly depends on both the influent C/N ratio and density of T. tubifex addition.

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The composition, herbicidal, larvicidal, and toxic activities of Juniperus horizontalis Moench essential oil and sabinene, its main component, were evaluated. The seed germination percentage and root length of eight different plant species (crops and weeds) were measured for in vitro herbicidal activity tests. Different doses (100, 50, 10, 5, 1, 0.5 µg/mL) of the samples were applied to seeds for 120 h. The same doses were applied to test the toxicity of the samples on Tubifex tubifex (sludge worm) and Chironomus aprilinus (blood worm). Four doses (435, 652.5, 870, and 1740) of samples were in a larvicidal test on Tenebrio molitor L. (mealworm), and bioassays were checked after 24 and 48 h. The analysis of the oil allowed for the identification of dominant components as sabinene (38.7%), α-pinene (10.0%), elemol (8.6%), γ-terpinene (8.3%), limonene (7.8%) and α-thujene (5.3%). The results showed that the effect of oil on root length inhibition was significant in all crop species. The doses which affected root growth were not toxic to Tubifex tubifex and Chironomus aprilinus. Finally, the obtained results in the larvicidal bioassay indicate that the potential of J. horizontalis in yellow mealworm survivorship limitation can be a starting point for future research.

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Urbanization and population growth demand the construction of structures to facilitate the need for space, and old infrastructures must make space for new ones leading to demolition and concrete debris. In addition to demolition, aging and weather are factors leading to concrete deterioration and, thus, a new challenge as an environmental pollutant. Studies on how concrete debris and leachate affect biota in the environment are limited. The present study aimed to understand the effects of leachate from various sizes of concrete debris on the three oligochaete species Enchytraeus crypticus, Tubifex, and Lumbriculus variegatus. Acute toxicity testing was carried out to determine the adverse effects over time. The oligochaetes' survival was monitored as well as the activity of the biotransformation enzyme glutathione S-transferase and the antioxidative enzyme catalase as indicators of the oxidative stress status. Leachate from the smallest concrete particle size (<1 mm) was found to be the most toxic as it caused, on average, 6-fold increased oligochaete mortality compared to the larger pieces (2-5 cm) after 96 h of exposure, potentially due to the larger surface area facilitating the release of toxicants. Substrate buffered the toxic effect of the leachate with 42 ± 12% fewer mortalities and reduced adverse effects on the enzymes. Of the three oligochaetes, E. crypticus was the most resilient to the concrete leachate. The study is the first to investigate the effects of concrete leachate on oligochaetes.

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The complete mitochondrial genome of Tubifex tubifex was analyzed using the MGISEQ-2000 platform. The size of the complete mitochondrial genome was 15,972 bp. Data pertaining to the genome, such as the presence of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and a putative control region were submitted to NCBI (MW690579). A phylogenetic tree was constructed with the sequences of the 13 PCGs using the maximum-likelihood method. Despite only a few references available on the complete mitochondrial genome of other aquatic oligochaetes, our phylogenetic analysis revealed that the freshwater oligochaetes T. tubifex and Limnodrilus hoffmeisteri are in a cluster different from that of the earthworm group.

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The study goal was to examine the effects of sand and mud on the propagation of Myxobolus cerebralis, the whirling disease agent, in four mitochondrial 16S ribosomal DNA lineages (I, III, V, VI) of its oligochaete host, Tubifex tubifex (Tt). In all the lineage groups held continuously in either substrate (non-shifted) or transferred from sand to mud (shifted), substrate influenced parasite proliferation only in lineage III. Sporogenesis and release of triactinomyxon spores (TAMs) were more prevalent in lineage III Tt in mud compared to sand. Low-infection prevalence and lack of parasite development in lineage I is associated with the greater number of resistant worms and were not affected by substrate type. Substrate did not impact Tt from lineages V and VI that failed to develop any parasite stages in either substrate even after shifting from sand to mud. The relationship between the microbial community in the substrate and parasite proliferation in lineage III was described but not analyzed due to small sample size. Substrate-associated bacteria were hypothesized as essential dietary source for the oligochaete host feeding selectively on fine (mud)-microflora. Progeny was produced by all lineage groups shifted to mud with disparate survival profiles in lineage V and VI and high mortalities in lineage III. Our study demonstrates that substrate type can alter parasite proliferation in lineage III. Conversely, parasite development and infectivity were not altered in lineage V and VI that are refractory to the parasite nor among the more resistant phenotypes (I), regardless of substrate type.

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Pollution levels of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) were investigated in the El Sol and the La Luna alpine lakes. The lakes are located in central Mexico, in the crater of the Nevado de Toluca volcano. The El Sol and the La Luna lakes are extremely relevant in Mexico and in the world because they are recognized as pristine regions and environmental reservoirs. Samples of atmospheric aerosol, sediment, plankton, and Tubifex tubifex (sludge worm) were collected at three different sample locations for three years (2017, 2018, and 2019) at three different times of year, meaning that the weather conditions at the time of sampling were different. Pollutants were analysed by gas chromatography-mass spectrometry with negative chemical ionisation (GC-MS/NCI). Endosulfan was the most frequent and abundant pollutant, showing the highest peaks of all. Atmospheric aerosol revealed Σ2 = 45 pg/m3, including α and ß, while sediment lakes displayed α, ß and endosulfan sulfate as Σ3 = 1963 pg/g, whereas plankton and Tubifex tubifex showed Σ2 = 576 pg/g and 540 pg/g for α and ß respectively. Results of endosulfan ratios (α/ß) and (α-ß/endosulfan sulfate) suggest that both fresh and old discharges continue to arrive at the lakes. This study shows for the first time the pollution levels of OCP and PCB in high mountain lakes in Mexico. These results that must be considered by policy makers to mitigate their use in the various productive activities of the region.

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Lygodium microphyllum Cav. (Family: Lygodiaceae) is a perennial, snake fern and an invasive weed in Florida and also known as old world climbing fern. This study is intended to evaluate the antipyretic, analgesic, anti-inflammatory, antidiarrheal and anthelmintic activity of methanol extract of Lygodium microphyllum Cav. leaves (MELM) by in vivo, in vitro and in silico approaches. In addition, Biovia, PyRx autoDock Vina, UCSF Chimera have been applied to investigate the docking study in order to evaluate the binding interaction and an online tool was used to explore the ADME/T properties of selected bioactive compounds. In acetic acid induced writhing study, MELM inhibited 44.28% and 56.61% of writhes at 200 and 400 (mg/kg) respectively compared to standard drug Diclofenac-Na (10 mg/kg) (74.42% inhibition). In anti-inflammatory experiment by formalin triggered licking method, MELM caused significant (p < 0.05) inhibition of licking in both early phase (42.97%, 63.30%) and late phase (43.35%, 63.03%) at the doses of 200 and 400 mg/kg respectively, whereas reference drug Ibuprofen inhibited paw licking 77.18% in early phase and 76.86% in late phase. MELM also showed promising antipyretic potential where the maximum reduction of fever was produced by MELM 400 mg/kg whose fever lowering capacity is close to the prescribe drug Indomethacin 4 mg/kg, i.p. In Castor oil triggered diarrhea method, MELM delayed the onset time of diarrhea, continuous persistence of wet feces, and decreased the weight of wet feces remarkably. Defection inhibition was achieved 27.56% and 51.72%, for MELM 200 and at 400 (mg/kg) respectively while loperamide 2 (mg/kg) yields 55.17% inhibition of the diarrheal defecation. In anthelmintic bioassay, MELM took 5.83 ± 0.83 and 41.67 ± 1.78 min respectively for paralyzing and death compared to standard drug albendazole; (paralysis time 4.00 ± 0.73 min and death time 31,00 ± 1.71 min). Isoeleutherol, isoquercetin and quercetin were found prominent in molecular docking study and ADME/T analysis verified their drug likeliness. The research validates the moderate analgesic, anti-inflammatory, and remarkable antipyretic, antidiarrheal, anthelmintic activities of the plant extract which can be used an alternative source of novel therapeutics.

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Metalaxyl and Metalaxyl-M are the fungicides that widely used in many countries. In this study, the environmental behaviors between metalaxyl and metalaxyl-M in Tubifex tubifex (T. tubifex) were quantitative analyzed by using a high performance liquid chromatography with photo-diode-array-detector (HPLC-DAD). Results demonstrated that there was no significant difference (p > 0.05) in the concentration of metalaxyl and metalaxyl-M in T. tubifex during the exposure process. However, the dissipation behaviors of metalaxyl and metalaxyl-M in T. tubifex were different (p < 0.05) during the non-exposure culture process. Meanwhile, the toxic effects were also evaluated by comparing the different influences of these two compounds on related physiological indicators, and functional enzyme activities. The survival rates of T. tubifex were 63.33 ± 15.28% (20 mg L-1), 63.33 ± 5.77% (200 mg L-1) treated with metalaxyl and were 50.00 ± 10.00% (20 mg L-1), 46.67 ± 11.55 (200 mg L-1) treated with metalaxyl-M at the non-exposure culture process. The autotomy rates were increased significantly compared with the initial in all treatments. Besides, the activities of CAT, SOD, and GST in T. tubifex were also inhibited by metalaxyl and metalaxyl-M treatments. Finally, the high-throughput transcriptome sequencing technology was applied to investigate the metabolic pathways of target analytes in T. tubifex, and results proved that the metabolic pathways associated with human diseases (such as viral myocarditis) were up-regulated expression for metalaxyl and metalaxyl-M treatments, and metalaxyl-M up-regulated more significantly. All the results demonstrated that metalaxyl-M had a higher toxicity than metalaxyl on T. tubifex.

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Sediment remediation in eutrophic aquatic ecosystems is imperative, but effective ecological measures are scarce. A pilot-scale trial investigated sediment remediation by the addition of Tubifex tubifex. The results showed that the addition of T. tubifex accelerated sediment organic matter (OM) and nitrogen (N) loss, with averages of 7.7% and 75.1% increased loss (IL) compared to treatments without T. tubifex in the 60-day experiment, respectively. The percentages of the increased in water to the IL in sediment were only 0.6%, 0.21%, 2.1% and 6.3% for NH4+-N, NOx--N, TN and COD, respectively, at the end of the experiment. The absolute abundances of the nitrifying genes AOA and AOB; the denitrifying genes napA, nirS, nirK, cnorB and nosZ; and the anaerobic ammonia oxidation gene anammox increased 2.3- to 11.0-fold with the addition of T. tubifex. Therefore, the addition of T. tubifex is an effective strategy for sediment remediation by accelerating OM and N loss in sediment without substantially increasing the water N concentration.

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The present study was aimed to assess the acute toxicity of organophosphate pesticide, profenofos; synthetic pyrethroid pesticide, λ cyhalothrin and biopesticide, azadirachtin and their sublethal effects on growth rate and oxidative stress biomarkers in Tubifex tubifex in vivo. The results showed that 96 h LC50 value of profenofos, λ cyhalothrin and azadirachtin to Tubifex tubifex are 0.59, 0.13 and 82.15 mg L-1 respectively. Pesticide treated worms showed several behavioral abnormalities including increased mucus secretion, erratic movements, wrinkling activity and decreased clumping tendency during acute exposure. The percentage of autotomy increased significantly (p < 0.05) with the increasing concentration of the pesticides at 96 h of exposure. Sublethal concentrations of profenofos (0.059 and 0.118 mg L-1), λ cyhalothrin (0.013 and 0.026 mg L-1) and azadirachtin (8.2 and 16.4 mg L-1) caused significant alterations in growth rate and oxidative stress enzymes in T. tubifex during 14 days exposure period. The growth rate of the pesticide exposed worms decreased significantly (P < 0.05) in a concentration and duration-dependent manner. Superoxide dismutase (SOD), reduced glutathione (GSH), glutathione-s-transferase (GST) and glutathione peroxidase (GPx) demonstrated a noteworthy (p < 0.05) initial induction followed by a subsequent reduction, while catalase (CAT) and malondialdehyde (MDA) exhibited noteworthy induction (p < 0.05) all through the exposure time. Through principal component analysis, correlation matrix, and integrated biomarker response, the effects of profenofos, λ cyhalothrin and azadirachtin on T. tubifex were distinguished. These results indicate that exposure to profenofos, λ cyhalothrin and azadirachtin affect survivability, change the behavioral responses, reduce the growth rate and induce oxidative stress enzymes in T. tubifex.

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Pilot-scale saturated vertical flow constructed wetlands (VF-CWs) were established to identify whether T. tubifex has the similar performance in saturated VF-CWs to that in surface flow CWs in improving pollutant removal efficiency (RE). The saturated VF-CWs with T. tubifex achieved REs of 67.3% total nitrogen (TN) and 39.8% chemical oxygen demand (COD), which were significantly higher than treatments without T. tubifex (42.2% TN and 31.4% COD). There existed significant interactions between macrophytes and T. tubifex. T. tubifex greatly improved the dissolved oxygen by increasing the connectivity between layers, and enhanced dehydrogenase activity and fluorescein diacetate. Adding T. tubifex improved the bacterial diversity and relative abundance of both N-cycle bacteria and fermentation bacteria in the biofilms. The improvements of ammonia oxidation and anammox were the main pathways for the increased nitrogen removal by T. tubifex. Therefore, T. tubifex is a useful tool for improving pollutant REs in saturated VF-CWs.

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The role of Tubifex tubifex in organic matter (OM) decomposition in aquatic ecosystems has been widely studied, but considerable uncertainties exist in terms of the effect mechanism. The effect of T. tubifex on sediment OM decomposition in laboratory-scale microcosms was quantified, and possible pathways were identified. In the first 7 days of the decomposition of OM mixed in sediment, no significant effect of T. tubifex on organic matter loss (OML) was observed for both low- and high-OM treatments; meanwhile, from day 7-60, T. tubifex addition significantly improved OML from 55.0%-57.5% to 71.8%-77.7% in the low-OM treatments and from 55.5%-56.6% to 64.1%-68.7% in the high-OM treatments. The enhanced OML observed with T. tubifex was mainly due to the promoted decomposition of refractory organic components, e.g., cellulose, hemicellulose and lignin. The proportion of refractory components in the gut of T. tubifex was significantly lower than that in the sediments (p < 0.01), indicating a pathway corresponding to the ingestion and digestion of refractory components by T. tubifex. Although T. tubifex reduced the water dissolved oxygen (DO) by increasing the water chemical oxygen demand (COD), the oxygen supply was improved by T. tubifex, and this could be affected by the increase in the relative abundance of aerobic to anaerobic bacteria in the sediments. T. tubifex significantly increased the diversity of the bacterial and fungal communities in the sediments. Moreover, the community structure of bacteria and fungi was substantially different between gut and sediment. Therefore, multiple pathways of the effect of T. tubifex on OM decomposition were established, and the results have great significance for the artificial manipulation of OM circulation using T. tubifex and the restoration of damaged aquatic ecosystems.

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Microplastics (MPs) are emerging pollutants, which are considered ubiquitous in aquatic ecosystems. The effects of MPs on aquatic biota are still poorly understood, and consequently, there is a need to understand the impacts that MPs may pose to organisms. In the present study, Tubifex tubifex, a freshwater oligochaete commonly used as a bioindicator of the aquatic environment, was exposed to fluorescent polyethylene microspheres (up to 10 µm in size) to test whether the oxidative stress status was affected. The mortality rate of T. tubifex, as well as the activities of the oxidative stress status biomarker enzymes glutathione reductase and peroxidase, were assessed. In terms of oxidative stress, no significant differences between the exposure organisms and the corresponding controls were detected. Even though the data suggest that polyethylene MPs and the selected concentrations did not pose a critical risk to T. tubifex, the previously reported tolerance of T. tubifex to environmental pollution should be taken into account and thus MPs as aquatic pollutants could still represent a threat to more sensitive oligochetes.

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Hexachlorocyclohexane isomers (HCHs) are persistent organic pollutants (POPs), having potential risks to humans and ecosystem. This work evaluated the propensity of organisms to accumulate, eliminate, and transfer HCHs along the food chain (Tubifex tubifex and common carp (Cyprinus carpio)). The accumulation of HCHs from water by worms and carp was observed, and the concentrations increased with exposure time. After 8 days, the HCH concentrations in organisms remained stable. The accumulation factor (AF) values of HCHs in T. tubifex were higher than those in carp, indicating that the bioaccumulation abilities of HCHs in T. tubifex were higher than those in carp. The contaminated worms as a dietary source in the food chain led to significantly higher bioaccumulation in carp. The biomagnification factor (BMF) values of HCH isomers were all greater than 1. In the dissipation experiments, the elimination was fast and the half-lives were shorter than 2.5 days. The enantioselective accumulation and dissipation of α-HCH enantiomers were observed in worms and carp (food chain), and the enantiomeric differences should be taken into consideration in the study of contaminants risk assessment. The results on trophic transfer of HCHs in a freshwater food chain should be helpful for better understanding the fate, transport, and transfer of HCHs in freshwater environments.

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Risk management of toxic substances is often based on Environmental Quality Standards (EQS) set for the water compartment, assuming they will also protect benthic organisms. In the absence of experimental data, EQS for sediments can be estimated by the equilibrium partitioning approach. The present study investigates whether this approach is protective of benthic organisms against pentachlorophenol (PCP), a legacy contaminant and EU priority substance still used in some parts of the world. Three freshwater species of invertebrates with different life cycles and feeding behaviors (the oligochaetes Lumbriculus variegatus, Tubifex tubifex and the dipteran insect Chironomus riparius) were exposed to PCP spiked sediments (2.10-46.03mgPCP/kg d.w. plus controls) in laboratory standard tests. Exposure duration was 28days for T. tubifex and L. variegatus and 10 and 28days for C. riparius; according to the corresponding OECD guidelines. For each investigated end-point, dose-response data were normalized to the mean control and fitted to a four-parameter log-logistic model for calculating the corresponding EC50 and EC10. The ranges for EC50 and EC10 estimates were 4.39 (Chironomus riparius-emergence)-27.50 (Tubifex tubifex-cocoon) and 0.30 (T. tubifex-young worms) -16.70 (T. tubifex-cocoon) mg/kg d.w., respectively. The EC50 and the EC10 values of L. variegatus were within these ranges. Following the EU Technical Guidance for deriving EQS, the lowest EC10 value of 0.30mg/kg (T. tubifex-young worms) resulted in a PCP quality standard (QS) for sediments of 30ng/g, about one fourth of the tentative QS of 119ng/g estimated by the equilibrium partitioning (EqP) approach. The response of benthic biota to PCP varied across organisms and across end-points for the same organism, so that the use of sediment PCP-QS calculated using the EqP-approach may be under-protective of the most sensitive organisms. Information on the possible effects of PCP on resident organisms must therefore be collected for appropriately managing aquatic systems.

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Early embryos of the clitellate annelid Tubifex (oligochaete) undergo a series of unequal spiral cell divisions before the descendants of the D quadrant micromeres (cells 2d and 4d) divide bilaterally. Here, we show that inhibition of zygotic transcription by microinjection of α-amanitin (transcription inhibitor) exclusively converts unequal cleavage in cell 2d11 (granddaughter of 2d) into equal cleavage while other unequal cleavages and ensuing bilateral cleavages in cells 4d and 2d111 (great-granddaughter of 2d) all proceed in a normal fashion in the presence of this inhibitor. These results differ significantly from those reported for embryos of another clitellate annelid Helobdella (leech), in which inhibition of transcription converts bilateral (symmetric) cleavages in cells DNOPQ"' and DM" (equivalent to 2d111 and 4d) into unequal (asymmetric) cleavages while having no apparent effect on unequal cleavage in DNOPQ" (equivalent to 2d11). These differences imply distinct mechanisms for the control of the unequal-to-bilateral transition in the two clitellate annelids.

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