Vibrational spectroscopic detection of radiation-induced structural changes in Chironomus hemoglobin.

Purpose: Chironomus hemoglobin is known to exhibit higher gamma radiation resistance compared to human hemoglobin. In the present study, we have introduced a sensitive method to analyze radiation-induced alterations in Chironomus hemoglobin using Vibrational spectroscopy and further highlighting its potential for monitoring radiotoxicity in aquatic environments. Materials and methods: Vibrational spectroscopic methods such as Raman and FT-IR spectroscopy were used to capture the distinctive chemical signature of Chironomus hemoglobin (ChHb) under both in vitro and in vivo conditions. Any radiation dose-dependent shifts could be analyzed Human hemoglobin (HuHb) as standard reference. Results: Distinctive Raman peak detected at 930 cm-1 in (ChHb) was attributed to C-N stretching in the heterocyclic ring surrounding the iron atom, preventing heme degradation even after exposure to 2400 Gy dose. In contrast, for (HuHb), the transition from deoxy-hemoglobin to met-hemoglobin at 1210 cm-1 indicated a disruption in oxygen binding after exposure to 1200 Gy dose. Furthermore, while ChHb exhibited a consistent peak at 1652 cm-1 in FT-IR analysis, HuHb on the other hand, suffered damage after gamma irradiation. Conclusion: The findings suggest that vibrational spectroscopic methods hold significant potential as a sensitive tool for detecting radiation-induced molecular alterations and damages. Chironomus hemoglobin, with its robust interaction of the pyrrole ring with Fe, serves as a reliable bioindicator molecule to detect radiation damage using vibrational spectroscopic method.

Improving Sediment Toxicity Testing for Very Hydrophobic Chemicals: Part 2-Exposure Duration, Upper Limit Test Concentrations, and Distinguishing Actual Toxicity from Physical Effects.

Sediment toxicity testing with very hydrophobic organic chemicals (VHOCs) is challenging because of the chemicals' low aqueous solubilities and slow kinetics. The present study presents the results of experiments investigating whether the standard exposure duration of 28 days with benthic invertebrates is sufficient for VHOCs; above which concentrations in sediment VHOCs are present as "free phase," that is, crystals or non-aqueous-phase liquids (NAPLs); and whether it is possible to discriminate between actual VHOC toxicity and physical effects caused by NAPLs through fouling of the test organisms. The results suggest that the standard sediment toxicity test duration is sufficient for obtaining steady-state VHOC concentrations in Hyalella azteca and Lumbriculus variegatus, provided that spiking and equilibration are performed properly (i.e., no free phase present). Under these conditions, transient (days 3-20) peak-shaped toxicokinetics were observed, with steady-state concentrations reached at approximately 28 days. The concentration above which NAPLs are present, the so-called critical separate phase concentration (CSPC), was determined for several VHOCs by modeling and two experimental methods. Modeling resulted in unrealistic and variable data and therefore should be applied with caution. Experimentally determining CSPCs was successful and yielded values of approximately 1000 (400-2000) mg/kg dry weight, depending on the chemical. Finally, it was demonstrated that distinguishing actual toxicity from physical effects is possible by applying a well-considered test setup, combining toxicity tests with multiple invertebrates (including Lumbriculus, which serves as a negative control for fouling); a broad test concentration range, preferably up to at least 30 000 mg/kg; and passive sampling to localize the CSPC. Applying this setup, false-positive effects due to fouling, as well as false-negative results due to testing at too low concentrations (trying to stay below the CSPC), can be avoided. Environ Toxicol Chem 2024;00:1-12. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effect of Aluminum Concentration in Water on Its Toxicity and Bioaccumulation in Zooplankton (Chaoborus and Chironomus) and Carp (Cyprinus carpio L.) Roe.

An attempt to evaluate aluminum toxicity to living organisms was undertaken in the study. A laboratory experiment was conducted to determine aluminum bioaccumulation and toxicity in Chironomus and Chaoborus larvae and in Cyprinus carpio L. roe depending on aluminum concentration in water reflecting natural chemical composition. Water was examined for temperature, pH, electrical conductivity, dissolved oxygen, color, nitrate nitrogen, nitrite nitrogen, sulfates by spectrophotometric method; total hardness and chlorides by titration method; and calcium, magnesium, sodium by flame atomic absorption spectrometry, total aluminum by electrothermal atomic absorption spectrometry. Determination of aluminum levels in water, roe, and zooplankton was carried out after mineralization using electrothermal atomic absorption spectrometry. Aluminum bioaccumulation factor in roe was determined with respect to concentration in water. Moreover, acute toxicity (LC50) was calculated. In the roe experiment, aluminum concentration in water at the end of the experiment was 0.0635-0.1283 mgAl∙dm-3. The lowest values were noted for the control sample and the highest for water with 0.03 mgAl∙dm-3 aluminum content. The final aluminum level in roe was, like in water, the highest in roe treated with 0.03 mgAl∙dm-3 (18.95 mgAl∙kg-1), and the lowest in roe treated with 3.00 mgAl∙dm-3 (6.96 mgAl∙kg-1). Aluminum bioaccumulation in roe was the strongest in the control. Survival rate ranged from 2.00 to 97.00%, which shows higher sensitivity of roe to aluminum concentration. LC50 value for Chaoborus was 0.6464 mgAl⋅dm-3, and for Chironomus 0.2076 mgAl⋅dm-3 indicating that Chironomus is more sensitive to aluminum toxic effects. Concentration of 3.0 mgAl∙dm-3 caused the highest mortality. Aluminum in both species at each concentration reached the highest levels after one day (24 h), 254.58 mg⋅kg-1 for Chaoborus and 3107 mg⋅kg-1 for Chironomus. After another day, aluminum levels decreased. This demonstrated the differential accumulation of aluminum depending on the species, which predisposes Chironomus as a better indicator organism. This type of research is important from the point of view of aquaculture, which is a targeted activity with a high degree of economic importance, but is also important for aquatic organisms living in natural conditions. Fish reproduction takes place in both types of waters, so all these reservoirs regardless of their source of destination should be inspected.

Combining Passive Sampling and Dosing to Unravel the Contribution of Hydrophobic Organic Contaminants to Sediment Ecotoxicity.

Contaminated sediments are ubiquitous repositories of pollutants and cause substantial environmental risks. Results of sediment bioassays remain difficult to interpret, however, as observed effects may be caused by a variety of (un)known stressors. This study aimed therefore to isolate the effects of hydrophobic organic contaminants from other (non)chemical stressors present in contaminated sediments, by employing a newly developed passive sampling-passive dosing (PSPD) test. The results showed that equilibrium partitioning between pesticides or polyaromatic hydrocarbons (PAHs) in contaminated sediments and a silicone rubber (SR) passive sampler was achieved after 1-3 days. Chlorpyrifos concentrations in pore water of spiked sediment matched very well with concentrations released from the SR into an aqueous test medium, showing that SR can serve as a passive dosing device. Subjecting the 96 h PSPD laboratory bioassay with nonbiting midge (Chironomus riparius) larvae to field-collected sediments showed that at two locations, concentrations of the hydrophobic organic contaminant mixtures were high enough to affect the test organisms. In conclusion, the developed PSPD test was able to isolate the effects of hydrophobic organic contaminants and provides a promising simplified building block for a suite of PSPD tests that after further validation could be used to unravel the contribution of hydrophobic organic chemicals to sediment ecotoxicity.

Species-specific Microorganisms in Acid-tolerant Chironomus Larvae Reared in a Neutral pH Range under Laboratory Conditions: Single Dataset Analysis.

To obtain a more detailed understanding of organismal acid tolerance, the larval microbiomes of 11 Chironomus species collected from acidic or neutral pH areas in Japan and reared at pH 7-8 under laboratory conditions were systematically compared using an amplicon sequencing ana-lysis. Evenness values were lower for the larval microbiomes of acid-tolerant Chironomus cf. riparius, Chironomus fusciceps, and Chironomus sulfurosus than for eight acid-sensitive species based on an alpha diversity ana-lysis. The lower evenness observed suggested a biased abundance of microorganisms, which was consistent with the identification of Chironomus species-specific microorganisms (such as Agromyces mediolanus and Comamonas odontotermitis related bacteria) with high abundance in acid-tolerant larvae. The abundance of specific microorganisms was also high in the microbiome of acid-tolerant larvae of Chironomus acerbiphilus reared at pH 4, but not in that of acid-sensitive larvae. Based on a PICRUSt2 ana-lysis, genes involved in saccharide transport were less abundant in the microbiome of acid-tolerant larvae than in that of acid-sensitive larvae, indicating nutrient-poor acidic environments. Although these results were obtained from single datasets, acid-tolerant larvae appeared to establish Chironomus species-specific interactions with microorganisms independent of saccharides, in contrast to acid-sensitive larvae. The present study is the first step towards understanding organismal acid tolerance.

Seasonal variations of microplastic in sediment, Chironomus sp. larvae, and chironomid tubes in two wastewater sites in Sohag Governorate, Egypt.

Microplastic (MP) contamination is an acknowledged global problem that poses a severe risk to aquatic ecosystem biota. Nevertheless, little is known about their prevalence in animal construction. The main objective of our study was to reduce the gap information of seasonal abundance, distribution, composition, and risk assessment of MP contamination. The concentrations of MPs in sediment, Chironomus sp. larvae, and their tubes were found to be higher in site 2 (S2) than in site 1 (S1) during the four seasons of the year. However, MP concentrations ranged from 312 ± 64.7 to 470 ± 70 items/kg dry weight, 0.79 ± 0.16 to 1.1 ± 0.3 particles/individual, and 0.5 ± 0.04 to 0.9 ± 0.04 particles/tube in sediment, Chironomus, and chironomid tubes, respectively. Blue and red polyester fibers are the most dominant MPs which are distributed in sediment, Chironomus, and chironomid tubes. The length of the dominant fiber accumulates in Chironomus, and their tubes are highly varied compared to that of the substrate. Additionally, we found that the mean number of MPs/individual larvae in the fourth instar was significantly higher than that in the second instar. Risk indicators for the environment, polymer risk assessment, and pollution load were estimated, where they were higher in S2 than in S1 correlated to MPs abundance and polymer type. The seasonal fluctuation in MP concentration, characterization, and risk in the two sites could depend on the amount of sewage effluent discharged into the wastewater treatment plants (WWTPs), which was reflected by Chironomus sp. larvae. Therefore, further research should be done to adopt the applicability of Chironomus as MP bioindicators in various freshwater environments throughout the world.

An ecological network approach for detecting and validating influential organisms for rice growth.

How to achieve sustainable food production while reducing environmental impacts is a major concern in agricultural science, and advanced breeding techniques are promising for achieving such goals. However, rice is usually grown under field conditions and influenced by surrounding ecological community members. How ecological communities influence the rice performance in the field has been underexplored despite the potential of ecological communities to establish an environment-friendly agricultural system. In the present study, we demonstrate an ecological-network-based approach to detect potentially influential, previously overlooked organisms for rice (Oryza sativa). First, we established small experimental rice plots, and measured rice growth and monitored ecological community dynamics intensively and extensively using quantitative environmental DNA metabarcoding in 2017 in Japan. We detected more than 1000 species (including microbes and macrobes such as insects) in the rice plots, and nonlinear time series analysis detected 52 potentially influential organisms with lower-level taxonomic information. The results of the time series analysis were validated under field conditions in 2019 by field manipulation experiments. In 2019, we focused on two species, Globisporangium nunn and Chironomus kiiensis, whose abundance was manipulated in artificial rice plots. The responses of rice, namely, the growth rate and gene expression patterns, were measured before and after the manipulation. We confirmed that, especially in the G. nunn-added treatment, rice growth rate and gene expression pattern were changed. In the present study, we demonstrated that intensive monitoring of an agricultural system and the application of nonlinear time series analysis were helpful to identify influential organisms under field conditions. Although the effects of the manipulations were relatively small, the research framework presented here has future potential to harness the ecological complexity and utilize it in agriculture. Our proof-of-concept study would be an important basis for the further development of field-basis system management.

UDP-glucuronosyltransferase is involved in susceptibility of Chironomus kiiensis Tokunaga, 1936 (Diptera: Chironomidae) to insecticides.

UDP-glucuronosyltransferases (UGTs) could transform various exogenous and endogenous compounds, which help detoxification of pesticides in insects. To investigate the role of UGTs in the detoxification metabolism of insecticides in Chironomus kiiensis, CkUGT302M1, CkUGT302N1, CkUGT308N1 and CkUGT36J1 genes were identified with 1449-1599 bp encoding 482-532 amino acids. Four UGT genes shared 40.86∼53.36% identity with other homologous insect species, and expressed in all developmental stages, notably in the larval and adult stages. Expression of CkUGTs was higher in the gastric caecum, midgut and head. Moreover, CkUGTs expression and activity were significantly increased in C. kiiensis larvae in exposure to sublethal concentrations of carbaryl, deltamethrin and phoxim, respectively. To further explore the functions of UGT genes, the CkUGT308N1 was effectively silenced in 4th instar C. kiiensis larvae by RNA interference, which resulted in the mortality of dsCkUGT308N1 treated larvae increased by 71.43%, 111.11% and 62.50% under sublethal doses of carbaryl, deltamethrin and phoxim at the 24-h time point, respectively. The study revealed that the CkUGT308N1 gene in C. kiiensis could contribute to the metabolism of pesticides and provide a scientific basis for evaluating the water pollution of pesticides.

Single-use take-away cups of paper are as toxic to aquatic midge larvae as plastic cups.

Single-use plastics and food packaging are the most common items polluting the environment, commonly identified in surveys and litter monitoring campaigns. There are pushes to ban these products from production and use in different regions, and to replace them with other materials viewed as "safer" or "more sustainable". Here, we address the potential environmental impacts of take-away cups and lids used for hot and cold beverages, consisting of plastic or paper. We produced leachates from plastic cups (polypropylene), lids (polystyrene), and paper cups (lined with polylactic acid), under conditions representative of plastic leaching in the environment. The packaging items were placed and left to leach in sediment and freshwater for up to four weeks, and we tested the toxicity of contaminated water and sediment separately. We used the model aquatic invertebrate Chironomus riparius and assessed multiple endpoints both on larval stages and on emergence to the adult phase. We observed a significant growth inhibition with all the materials tested when the larvae were exposed in contaminated sediment. Developmental delays were also observed for all materials, both in contaminated water and sediment. We investigated teratogenic effects via the analysis of mouthpart deformities in chironomid larvae, and observed significant effects on larvae exposed to polystyrene lid leachates (in sediment). Finally, a significant delay in time to emergence was observed for females exposed to paper cups leachates (in sediment). Overall, our results indicate that all the tested food packaging materials can have adverse effects on chironomids. These effects can be observed from one week of material leaching in environmental conditions, and tend to increase with increasing leaching time. Moreover, more effects were observed in contaminated sediment, indicating that benthic organisms might be especially at risk. This study highlights the risk posed by take-away packaging and their associated chemicals, once discarded into the environment.

Chironomus sp. as a Bioindicator for Assessing Microplastic Contamination and the Heavy Metals Associated with It in the Sediment of Wastewater in Sohag Governorate, Egypt.

The consequences of plastic waste pollution have imposed wide global concerns. One of these consequences is the production of micro- and nanosized particles (MNPLs) from aged plastics. The problem of MNPLs is magnified by their potential to transport various contaminants due to their large surface area and other variable physiochemical properties. From this point on, it is important to know the real concentration of MNPLs in our environment and their potential to internalize wild organisms as well as transfer contaminants that are completely highlighted. As a result, our study is the first to detect MP pollution in the upper Egypt wastewater environment. It could be utilized as a baseline to estimate MP wastes and develop management techniques, particularly in Sohag Governorate. The concentration and characterization of MPs in sludge, water, Chironomus sp. larvae, and their tubes were studied in this work. Chironomus sp. is a reliable bioindicator prevalent in such contaminated environments, and it was used to demonstrate how MPs invade biological barriers. Our results found that red and blue polyester fibers are much more prevalent than other polymers, colors, and shapes of MPs. While each dry kilogram of wastewater sludge contains 310 ± 84 particles, this amount is reduced to 1.55 ± 0.7 per liter in the water column. Biologically, the present study succeeded in detecting the MPs inside the wild organism, with concentrations reaching 71 ± 21 and 4.41 ± 1.1 particles per gram wet weight in Chironomus sp. larvae and their tubes (chironomid tubes), respectively. The potential hazard of MPs stems from their propensity to transfer pollutants. At this point, our findings revealed a corresponding and significant concentration of various heavy metals (Cu, Pb, Cd, and Ni) detected in MPs or Chironomus sp. versus sludge. In conclusion, our findings not only proved the presence of MPs in wastewater but also demonstrated their ability to internalize cross-wild organisms, allowing toxins to accumulate inside their bodies, raising concerns about the possible health impacts of plastic pollution.

Graphene Oxides (GOs) with Different Lateral Dimensions and Thicknesses Affect the Molecular Response in Chironomus riparius.

Graphene oxide (GO) materials possess physicochemical properties that facilitate their application in the industrial and medical sectors. The use of graphene may pose a threat to biota, especially aquatic life. In addition, the properties of nanomaterials can differentially affect cell and molecular responses. Therefore, it is essential to study and define the possible genotoxicity of GO materials to aquatic organisms and their ecosystems. In this study, we investigated the changes in the expression of 11 genes in the aquatic organism Chironomus riparius after 96 h of exposure to small GOs (sGO), large GOs (lGO) and monolayer GOs (mlGO) at 50, 500 and 3000 µg/L. Results showed that the different genes encoding heat shock proteins (hsp90, hsp70 and hsp27) were overexpressed after exposure to these nanomaterials. In addition, ATM and NLK-the genes involved in DNA repair mechanisms-were altered at the transcriptional level. DECAY, an apoptotic caspase, was only activated by larger size GO materials, mlGO and lGO. Finally, the gene encoding manganese superoxide dismutase (MnSOD) showed higher expression in the mlG O-treated larvae. The lGO and mlGO treatments indicated high mRNA levels of a developmental gene (FKBP39) and an endocrine pathway-related gene (DRONC). These two genes were only activated by the larger GO materials. The results indicate that larger and thicker GO nanomaterials alter the transcription of genes involved in cellular stress, oxidative stress, DNA damage, apoptosis, endocrine and development in C. riparius. This shows that various cellular processes are modified and affected, providing some of the first evidence for the action mechanisms of GOs in invertebrates. In short, the alterations produced by graphene materials should be further studied to evaluate their effect on the biota to show a more realistic scenario of what is happening at the molecular level.

Enlarging the Arsenal of Test Species for Sediment Quality Assessment.

Since only a few standard benthic test species are available for sediment quality, our study aimed to employ multiple test species representing different sensitivity categories in the quality assessment of contaminated sediments. To this end three macroinvertebrate species, Sericostoma personatum (caddisfly, sensitivity category 10), Asellus aquaticus (isopod, category 3) and Chironomus riparius (chironomid, category 2), were exposed to sediments originating from various contamination sources in whole sediment bioassays using intact sediment cores. The agricultural sediment caused insect mortality, the agricultural and urban sediment caused isopod growth reduction and the urban and Wastewater Treatment Plant (WWTP) sediment affected chironomid emergence time. It is concluded that the arsenal of standard species can be successfully expanded by non-standard species, reducing over- or underestimation of the risks of contaminated sediments.

Chironomus riparius Larval Gut Bacteriobiota and Its Potential in Microplastic Degradation.

Chironomus riparius are sediment-dwelling invertebrates in freshwater ecosystems and are used as indicators of environmental pollution. Their habitat is threatened by high levels of contaminants such as microplastics and organic matter. A promising strategy for the eco-friendly degradation of pollutants is the use of bacteria and their enzymatic activity. The aim of this study was to characterize for the first time bacteriobiota associated with the gut of C. riparius larvae from nature and laboratory samples, to compare it with sediment and food as potential sources of gut microbiota, and to assess its ability to degrade cellulose, proteins, and three different types of microplastics (polyethylene, polyvinyl chloride, and polyamide). The metabarcoding approach highlighted Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota as most abundant in both gut samples. Culturable microbiota analysis revealed Metabacillus idriensis, Peribacillus simplex, Neobacillus cucumis, Bacillus thuringiensis/toyonensis, and Fictibacillus phosphorivorans as five common species for nature and laboratory samples. Two P. simplex and one P. frigoritolerans isolates showed the ability for intensive growth on polyethylene, polyvinyl chloride, and polyamide. Both cellulolytic and proteolytic activity was observed for Paenibacillus xylanexedens and P. amylolyticus isolates. The characterized strains are promising candidates for the development of environmentally friendly strategies to degrade organic pollution and microplastics in freshwater ecosystems.

Metal Exposure and Sex Shape the Fatty Acid Profile of Midges and Reduce the Aquatic Subsidy to Terrestrial Food Webs.

Aquatic micropollutants can be transported to terrestrial systems and their consumers by emergent aquatic insects. However, micropollutants, such as metals, may also affect the flux of physiologically important polyunsaturated fatty acids (PUFAs). As certain PUFAs have been linked to physiological fitness and breeding success of terrestrial consumers, reduced fluxes from aquatic systems could affect terrestrial populations and food webs. We chronically exposed larvae of the aquatic insect Chironomus riparius to a range of environmentally relevant sediment contents of cadmium (Cd) or copper (Cu) in a 28-day microcosm study. Since elevated water temperatures can enhance metals' toxic effects, we used two temperature regimes, control and periodically elevated temperatures (heat waves) reflecting an aspect of climate change. Cd and Cu significantly reduced adult emergence by up to 95% and 45%, respectively, while elevated temperatures had negligible effects. Both metal contents were strongly reduced (∼90%) during metamorphosis. Furthermore, the chironomid FA profile was significantly altered during metamorphosis with the factors sex and metal exposure being relevant predictors. Consequently, fluxes of physiologically important PUFAs by emergent adults were reduced by up to ∼80%. Our results suggest that considering fluxes of physiologically important compounds, such as PUFAs, by emergent aquatic insects is important to understand the implications of aquatic micropollutants on aquatic-terrestrial meta-ecosystems.

Evaluating the Functional Equivalency of Test Organism Performance in Negative and Solvent Controls During Chronic Sediment Ecotoxicity Studies Based on US Environmental Protection Agency Guidance.

The US Environmental Protection Agency (USEPA) considers sediment toxicity tests as conditional registration requirements for pesticides with soil Kd ≥50 L/kg-solid, Koc ≥1000 L/kg-organic carbon, or log Kow ≥3. The hydrophobicity of these compounds often necessitates use of solvents to ensure accurate and homogeneous dosing of spiked-sediment studies. For sediment tests, a volatile solvent (e.g., acetone) is generally used as a transient carrier. Due to low water solubility, test material is dissolved in a volatile solvent to create stock solutions. A measured aliquot of stock solution is then mixed with sand substrate, after which the solvent is evaporated. This spiking process results in negligible solvent exposure to organisms. In 2016, USEPA released final ecotoxicity test guidelines for subchronic freshwater (850.1735) and marine (850.1740) sediment test. These methods provide an option for conducting experiments with only a solvent control and no negative control. To adopt this testing strategy, functional equivalency between the negative and solvent control must be demonstrated. These test guidelines describe specific factors that should be considered for evaluating functional equivalency, including (a) the concentration of solvent in the test sediment after evaporation, (b) the levels of solvent that are known to affect organism health, (c) the known impurities in the solvent and their potential impact on organism health, and (d) the historical organism performance of solvent versus negative controls. Our analysis considers these factors and overall supports the elimination of the negative control requirement because this change is unlikely to impact the robustness or interpretability of spiked-sediment toxicity tests. Environ Toxicol Chem 2023;00:1-7. © 2023 CropLife America. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Chironomus sancticaroli (Diptera: Chironomidae) in ecotoxicology: laboratory cultures and tests.

Chironomus sancticaroli is a tropical species, easy to grow and to maintain in laboratory cultures. It has a fast reproduction cycle, under adequate conditions, around 30 days, allowing it to have many generations per year, an important criterion for selecting a test organism in ecotoxicology. Its life stages include: eggs, four larval instars (one planktonic and three benthic), pupa and adult (midges) This study aimed to: (1) review the methods for C. sancticaroli cultivation and its use in ecotoxicological tests, (2) establish a laboratory culture of C. sancticaroli, presenting the difficulties and discussing the ways to overcome them. Early 4th instar larvae was the most used in acute studies, while the 1st instar larvae (early 1st instar) was the most used in chronic studies; 96 h and 28 days were the most frequent durations in acute and chronic studies, respectively. The most common endpoints evaluated were organisms' survival and development, and most of the ecotoxicological studies using C. sancticaroli were performed in laboratory. Most of the tested contaminants were pesticides and these had the most adverse effects on organisms. Most mesocosms with environmental contaminated samples did not show adverse effects on C. sancticaroli. Chronic and field studies as well as those testing the effects of the mixture contaminants on C. sancticaroli were still deficient. Keeping the laboratory environment and equipment effectively sanitized was important as well as maintaining stabilized conditions of temperature, photoperiod, physical, chemical and biological water quality in cultures.

Utilization of piperonyl butoxide and 1-aminobenzotriazole for metabolic studies of toxic chemicals in Daphnia magna and Chironomus yoshimatsui.

Daphnids and chironomids have been used to assess the ecological effects of chemicals released into water bodies; however, the toxicity mechanisms in organisms are generally difficult to identify. Here, we developed a system capable of estimating the contribution of cytochrome P450 (CYP) to the metabolism of test substances in Daphnia magna and Chironomus yoshimatsui based on toxicity differences in the absence and presence of the CYP inhibitors piperonyl butoxide (PBO) and 1-aminobenzotriazole (ABT). The optimum concentrations of PBO and ABT that could effectively reduce the toxicity of diazinon, which is toxic after oxidative metabolism in vivo, were determined as 0.5 and 0.6 mg/L for D. magna, and 2.0 and 40.0 mg/L for C. yoshimatsui, respectively. Acute immobilization tests of 15 insecticides were conducted for D. magna and C. yoshimatsui, with and without the optimum concentrations of PBO or ABT. In the presence of either inhibitor, chlorpyrifos and chlorfenapyr toxicity was reduced in both organisms, whereas those of thiocyclam, nereistoxin, and silafluofen were enhanced in C. yoshimatsui. Liquid chromatography-mass spectrometry analysis of D. magna and C. yoshimatsui samples exposed to chlorfenapyr confirmed that the level of the active metabolite produced by CYP was decreased by PBO or ABT in both organisms. The system to which the test substance was co-exposed to PBO or ABT will be valuable for estimating the contribution of CYPs to metabolism and elucidating the toxicity mechanism in daphnids and chironomids.

Toxicological effects caused by environmental relevant concentrations of ketoconazole in Chironomus sancticaroli (Diptera, Chironomidae) larvae evaluated by oxidative stress biomarkers.

Ketoconazole (KTZ), a broad-spectrum fungicidal drug, has been a significant problem in recent decades due to its toxic action on non-target aquatic organisms. Thus, the present study aimed to evaluate determine the effects that environmental relevant concentration of the commercial formulation of KTZ can exert on benthic macroinvertebrates, more specifically on larvae of the insect Chironomus sancticaroli. Acute toxicity tests with KTZ indicated lethal concentration (LC50) of 9.9 µg/L. Analyses of prolonged exposure to KTZ (chronic toxicity) indicated an increase in the rate of mentum deformity by approximately 3 times at concentrations of 0.6 and 2.4 µg/L. All biomarkers analyzed showed an increase after exposure to KTZ (0.6 and 2.4 µg/L), with average values of 115 % for superoxide dismutase (SOD), 63 % for catalase (CAT), 111 % for glutathione S-transferase (GST) and 59 % for malonaldehyde (MDA) in C. sancticaroli larvae. Thus, the toxic effects on survival, development (length and weight), mentum and redox responses caused by commercial KTZ in low concentrations were observed on C. sancticaroli larvae. In addition, the results suggest that biochemical biomarkers can be used for studies involving environmental disturbances.

Juniperus horizontalis Moench: Chemical Composition, Herbicidal and Insecticidal Activities of Its Essential Oil and of Its Main Component, Sabinene.

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.

Karyotypes and COI Gene Sequences of Chironomus sp. Le1 (Kiknadze and Salova, 1996), Ch. laetus (Belyanina and Filinkova, 1996) and Their Hybrid from the Yamal Peninsula, Arctic Zone of Russia.

The study of the biological diversity of the Arctic zone yields intriguing results. Initial research on the lakes of the Yamal Peninsula resulted in the identification of Chironomus laetus and the hybrid Ch. laetus × Ch. sp. Le1. To avoid misidentification, we used morphological, cytogenetic, and molecular genetic approaches. By cytogenetics, in Ch. sp. Le1, seven banding sequences were found: Le1A1, Le1B1, Le1C1, Le1D1, Le1E1, Le1F1, and Le1G1. The karyotype of Ch. laetus was mapped for the first time; it is the first species with the arm combinations AE BC DF G. We propose the name of a new cytocomplex-"laetus". DNA-barcoding of the COI gene was carried out for Ch. laetus and Ch. laetus × Ch. sp. Le1 for the first time. The estimated genetic distance between the sequences of Ch. laetus and Ch. riihimakiensis is 2.3-2.5%. The high similarity in morphology, banding sequences, and the possibility of hybridization indicate a close relationship between Ch. laetus and Ch. sp. Le1, which is assumed to be the northern variant of Ch. riihimakiensis. Molecular genetic data suggests the presence of a subgroup with Ch. laetus.