Metabolomic analysis of combined exposure to microplastics and methylmercury in the brackish water flea Diaphanosoma celebensis.

Owing to their widespread distribution and high bioaccumulation, microplastics (MPs) and mercury (Hg) are considered major threats to the ocean. MP interacts with Hg because of its high adsorption properties. However, their toxicological interactions with marine organisms, especially combined effects at the molecular level, are poorly understood. This study investigated the single and combined effects of MP and Hg on the metabolic profile of the brackish water flea Diaphanosoma celebensis. A total of 238 metabolites were significantly affected by MP, Hg, or MP + Hg. Metabolite perturbation patterns showed that toxicity of Hg and MP + Hg was similar and that of MP was not significant. Among the 223 metabolites affected by Hg, profiles of 32 unannotated metabolites were significantly different from those of MP + Hg, and combined effects of MP + Hg decreased the effect of Hg on 25 of these metabolites. Only 11 annotated metabolites were significantly affected by Hg or MP + Hg and were related to carbohydrate, lipid, vitamin, and ecdysteroid metabolism. Ten metabolites were decreased by Hg and MP + Hg and were not significantly different between the exposure groups. Enrichment analysis showed that galactose, starch, and sucrose metabolism were the most affected pathways. These findings suggest that MP has negligible toxic effect, and Hg can induce energy depletion, membrane damage, and disruption of growth, development, and reproduction. Although the impact of MP was negligible, the combined effects of MP + Hg could be metabolite specific. This study provides better understanding of the combined effects of MP and Hg on marine organisms.

Endocrine Disruptor Exposure Causes Infochemical Dysregulation and an Ecological Cascade from Zooplankton to Algae.

Endocrine disruption is intimately linked to controlling the population of pollutant-exposed organisms through reproduction and development dysregulation. This study investigated how endocrine disruption in a predator organism could affect prey species biology through infochemical communication. Daphnia magna and Chlorella vulgaris were chosen as model prey and predator planktons, respectively, and fenoxycarb was used for disrupting the endocrine system of D. magna. Hormones as well as endo- and exometabolomes were extracted from daphnids and algal cells and their culture media and analyzed using liquid chromatography with tandem mass spectrometry. Biomolecular perturbations of D. magna under impaired offspring production and hormone dysregulation were observed. Differential biomolecular responses of the prey C. vulgaris, indicating changes in methylation and infochemical communication, were subsequently observed under the exposure to predator culture media, containing infochemicals released from the reproducibly normal and abnormal D. magna, as results of fenoxycarb exposure. The observed cross-species transfer of the endocrine disruption consequences, initiated from D. magna, and mediated through infochemical communication, demonstrates a novel discovery and emphasizes the broader ecological risk of endocrine disruptors beyond reproduction disruption in target organisms.

Targeting the Lowest Concentration of a Toxin That Induces a Detectable Metabolic Response in Living Organisms: Time-Resolved In Vivo 2D NMR during a Concentration Ramp.

In vivo nuclear magnetic resonance (NMR) is a powerful analytical tool for probing complex biological processes inside living organisms. However, due to magnetic susceptibility broadening, which produces broad lines in one-dimensional NMR, 1H-13C two-dimensional (2D) NMR is required for metabolite monitoring in vivo. As each 2D experiment is time-consuming, often hours, this limits the temporal resolution over which in vivo processes can be monitored. Furthermore, to understand concentration-dependent responses, studies are traditionally repeated using different contaminant and toxin concentrations, which can make studies prohibitively long (potentially months). In this study, time-resolved non-uniform sampling NMR is performed in the presence of a contaminant concentration sweep. The result is that the lowest concentration that elicits a metabolic response can be rapidly detected, while the metabolic pathways impacted provide information about the toxic mode of action of the toxin. The lowest concentration of bisphenol A (BPA) that induces a response was ∼0.1 mg/L (detected in just 16 min), while changes in different metabolites suggest a complex multipathway response that leads to protein degradation at higher BPA concentrations. This proof of concept shows it is possible, on the basis of "real-time" organism responses, to identify the sublethal concentration at which a toxin impacts an organism and thus represents an essential analytical tool for the next generation of toxicity-based research and monitoring.

Understanding the Fate of Environmental Chemicals Inside Living Organisms: NMR-Based 13C Isotopic Suppression Selects Only the Molecule of Interest within 13C-Enriched Organisms.

In vivo nuclear magnetic resonance (NMR) is rapidly evolving as a critical tool as it offers real-time metabolic information, which is crucial for delineating complex toxic response pathways in living systems. Organisms such as Daphnia magna (water fleas) and Hyalella azteca (freshwater shrimps) are commonly 13C-enriched to increase the signal in NMR experiments. A key goal of in vivo NMR is to monitor how molecules (nutrients, contaminants, or drugs) are metabolized. Conventionally, these studies would normally involve using a 13C-enriched probe molecule and feeding this to an organism at natural abundance, in turn allowing the fate of the probe molecule to be selectively analyzed. The drawback of such an approach is that there is a limited range of 13C-enriched probe molecules, and if available, they are extremely cost prohibitive. Uniquely, when utilizing 13C organisms, a reverse strategy of isotopic filtering becomes possible. The concept described here uses 1H detection in combination with a 13C filter on living organisms. The purpose is to suppress all 1H signals from the organism (i.e., 1H attached to 13C), leaving only the probe molecule (1H attached to 12C). Because the probe molecule can be selectively observed using this approach, it then makes it possible to follow and discern processes such as bioconversion, bioaccumulation, and excretion in vivo. As the approach uses 1H detection, it provides excellent detection limits in the nanogram range. In this article, the approach is introduced, optimized on standards, and then applied to follow nicotine biotransformation and lipid assimilation in vivo to demonstrate the concept.

Reproduction Stage Differentiates the Time-Course Regulation of Metabolites in Daphnia magna.

Daphnia magna is a keystone indicator zooplankton used in environmental quality assessments. Comparative metabolomics, which contrasts small biomolecular regulations under different conditions, has emerged as a sensitive and informative investigation tool for the assessment of environmental stressors on D. magna. Baseline metabolomic variation is likely impacted by the asexual reproduction cycle of D. magna; however, the relationship between metabolite concentration regulation and reproduction cycle has not been investigated. This study investigated the time-course regulation of 51 metabolites during different reproduction stages to determine how the reproduction cycle controlled the metabolite profile of D. magna. Two-way analysis of variance (ANOVA) results reveal that most metabolites show significantly differentiated concentrations by individual or a combination of reproduction stages and sampling time. On the basis of the partial least-squares-discriminant analysis (PLS-DA) and ANOVA-simultaneous component analysis (ANOVA-SCA), stages 2 and 3 of reproduction show similarity in metabolite abundance regulation compared to stage 1. Metabolites were classified as being either dependent or independent of reproduction in the pairwise concentration correlation based on Pearson correlation coefficients. This study observed that the D. magna reproduction stage is an important consideration and potential variable and should be considered carefully when conducting metabolomic experiments using D. magna.

Sorption of Nitro Explosives to Polymer/Biomass-Derived Biochar.

Factors affecting the sorptive removal of nitro explosives (2,4,6-trinitrotoluene [TNT] and hexahydro-1,3,5-trinitro-1,3,5-triazine [RDX]) to polymer/biomass-derived biochar were investigated through batch experiments. Compared with that of rice ( L.) straw (RS)-derived biochar, the sorption of TNT and RDX to polymer/RS-derived biochar was greatly enhanced by >2.5 and 4 times, respectively. The type and amount of polymer did not significantly affect the sorption of nitro explosives to polymer/RS-derived biochar. Pyrolysis temperature did not affect the sorption capacity. Surface treatment with acid or an oxidant did not significantly change the sorption capacity, suggesting that polymer residues may be strongly responsible for the enhancement. Possible polymer residues were identified via gas chromatography mass spectrometry analysis. The toxicity characteristic leaching procedure and Microtox bioassay analyses indicated that polymer/RS-derived biochar did not show possible harmful effects. Our results suggest that polymer/RS-derived biochar can be effectively used as a sorbent to remove nitro explosives both in the natural environment and engineered systems.

Multigenerational Effects of the Antibiotic Tetracycline on Transcriptional Responses of Daphnia magna and Its Relationship to Higher Levels of Biological Organizations.

Given the risk of environmental pollution by pharmaceutical compounds and the effects of these compounds on exposed ecosystems, ecologically relevant and realistic assessments are required. However, many studies have been mostly focused on individual responses in a single generation exposed to one-effect concentrations. Here, transcriptional responses of the crustacean Daphnia magna to the antibiotic tetracycline across multiple generations and effect concentrations were investigated. The results demonstrated that tetracycline induced different transcriptional responses of daphnids that were dependent on dose and generation. For example, reproduction-related expressed sequence tags (ESTs), including vitellogenin, were distinctly related to the dose-dependent tetracycline exposure, whereas multigenerational exposure induced significant change of molting-related ESTs such as cuticle protein. A total of 65 ESTs were shared in all contrasts, suggesting a conserved mechanism of tetracycline toxicity regardless of exposure concentration or time. Most of them were associated with general stress responses including translation, protein and carbohydrate metabolism, and oxidative phosphorylation. In addition, effects across the dose-response curve showed higher correlative connections among transcriptional, physiological, and individual responses than multigenerational effects. In the multigenerational exposure, the connectivity between adjacent generations decreased with increasing generation number. The results clearly highlight that exposure concentration and time trigger different mechanisms and functions, providing further evidence that multigenerational and dose-response effects cannot be neglected in environmental risk assessment.

Sorption and toxicity reduction of pharmaceutically active compounds and endocrine disrupting chemicals in the presence of colloidal humic acid.

This study investigated the toxicity changes and sorption of pharmaceuticals and endocrine disrupters in the presence of humic acid (HA). For the sorption experiment, a dead end filtration (DEF) system was used to separate bound and free-form target compounds. An algae growth inhibition test and E-screen assay were conducted to estimate the toxic effect of pharmaceutically active compounds (PhACs) and endocrine disrupting chemicals (EDCs), respectively. The permeate concentration was confirmed using liquid chromatography-mass spectrometry. In the sorption test, we observed significant sorption of PhACs and EDCs on colloidal HA, except for sulfamethoxazole (SMX). The values of log KCOC derived from DEF determinations ranged from 4.40 to 5.03. The removal efficiency varied with the HA concentration and the target chemical properties. Tetracycline and 4-octylphenol showed the highest sorption or removal efficiency (≈50%), even at 5 mg C/L HA. The algal growth inhibition of PhACs and the estrogenic effects of EDCs were significantly decreased in proportion to HA concentrations, except for SMX. In addition, the chemical analysis results showed a positive relationship with the bioassay results. Consequently, the sorption of PhACs and EDCs onto colloidal HA should be emphasized in natural environments because it significantly reduces bioavailable concentrations and toxicity to aquatic organisms.

Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel Bastille-like group of phages in the subfamily Spounavirinae.

BACKGROUND: Spounavirinae viruses have received an increasing interest as tools for the control of harmful bacteria due to their relatively broad host range and strictly virulent phenotype. RESULTS: In this study, we collected and analyzed the complete genome sequences of 61 published phages, either ICTV-classified or candidate members of the Spounavirinae subfamily of the Myoviridae. A set of comparative analyses identified a distinct, recently proposed Bastille-like phage group within the Spounavirinae. More importantly, type 1 thymidylate synthase (TS1) and dihydrofolate reductase (DHFR) genes were shown to be unique for the members of the proposed Bastille-like phage group, and are suitable as molecular markers. We also show that the members of this group encode beta-lactamase and/or sporulation-related SpoIIIE homologs, possibly questioning their suitability as biocontrol agents. CONCLUSIONS: We confirm the creation of a new genus--the "Bastille-like group"--in Spounavirinae, and propose that the presence of TS1- and DHFR-encoding genes could serve as signatures for the new Bastille-like group. In addition, the presence of metallo-beta-lactamase and/or SpoIIIE homologs in all members of Bastille-like group phages makes questionable their suitability for use in biocontrol.

Complete genome sequence analysis and identification of putative metallo-beta-lactamase and SpoIIIE homologs in Bacillus cereus group phage BCP8-2, a new member of the proposed Bastille-like group.

Bacillus cereus group-specific bacteriophage BCP8-2 exhibits a broad lysis spectrum among food and human isolates (330/364) of B. cereus while not infecting B. subtilis (50) or B. licheniformis (12) strains. Its genome is 159,071 bp long with 220 open reading frames, including genes for putative methyltransferases, metallo-beta-lactamase, and a sporulation-related SpoIIIE homolog, as wells as 18 tRNAs. Comparative genome analysis showed that BCP8-2 is related to the recently proposed Bastille-like phages, but not with either SPO1-like or Twort-like phages of the subfamily Spounavirinae.

Machine learning-based water quality prediction using octennial in-situ Daphnia magna biological early warning system data.

Biological early warning system (BEWS) has been globally used for surface water quality monitoring. Despite its extensive use, BEWS has exhibited limitations, including difficulties in biological interpretation and low alarm reproducibility. This study addressed these issues by applying machine learning (ML) models to eight years of in-situ BEWS data for Daphnia magna. Six ML models were adopted to predict contamination alarms from Daphnia behavioral parameters. The light gradient boosting machine model demonstrated the most significant improvement in predicting alarms from Daphnia behaviors. Compared with the traditional BEWS alarm index, the ML model enhanced the precision and recall by 29.50% and 43.41%, respectively. The speed distribution index and swimming speed were significant parameters for predicting water quality warnings. The nonlinear relationships between the monitored Daphnia behaviors and water physicochemical water quality parameters (i.e., flow rate, Chlorophyll-a concentration, water temperature, and conductivity) were identified by ML models for simulating Daphnia behavior based on the water contaminants. These findings suggest that ML models have the potential to establish a robust framework for advancing the predictive capabilities of BEWS, providing a promising avenue for real-time and accurate assessment of water quality. Thereby, it can contribute to more proactive and effective water quality management strategies.

Fungal Microbiome of Alive and Dead Korean Fir in its Native Habitats.

A rapid decline of Abies koreana has been reported in most of the natural alpine habitats in Korea. It is generally accepted that this phenomenon is due to climate change even though no clear conclusions have been drawn. Most research has focused on abiotic environmental factors, but studies on the relationships between A. koreana and soil fungal microbiomes are scarce. In this study, the rhizoplane and rhizosphere fungal communities in the alive and dead Korean fir trees from its three major natural habitats including Mt. Deogyu, Mt. Halla, and Mt. Jiri in Korea were investigated to identify specific soil fungal groups closely associated with A. koreana. Soil fungal diversity in each study site was significantly different from another based on the beta diversity calculations. Heat tree analysis at the genus level showed that Clavulina, Beauveria, and Tomentella were most abundant in the healthy trees probably by forming ectomycorrhizae with Korean fir growth and controlling pests and diseases. However, Calocera, Dacrymyces, Gyoerffyella, Hydnotrya, Microdochium, Hyaloscypha, Mycosymbioces, and Podospora were abundant in the dead trees. Our findings suggested that Clavulina, Beauveria, and Tomentella are the major players that could be considered in future reforestation programs to establish ectomycorrhizal networks and promote growth. These genera may have played a significant role in the survival and growth of A. koreana in its natural habitats. In particular, the genus Gyoerffyella may account for the death of the seedlings. Our work presented exploratory research on the specific fungal taxa associated with the status of A. koreana.

Toxic effects of fragmented polyethylene terephthalate particles on the marine rotifer Brachionus koreanus: Based on ingestion and egestion assay, in vivo toxicity test, and multi-omics analysis.

Microplastics (MPs) are a major concern in marine ecosystem because MPs are persistent and ubiquitous in oceans and are easily consumed by marine biota. Although many studies have reported the toxicity of MPs to marine biota, the toxicity of environmentally relevant types of MPs is little understood. We investigated the toxic effects of fragmented polyethylene terephthalate (PET) MP, one of the most abundant MPs in the ocean, on the marine rotifer Brachionus koreanus at the individual and molecular level. No significant rotifer mortality was observed after exposure to PET MPs for 24 and 48 h. The ingestion and egestion assays showed that rotifers readily ingested PET MPs in the absence of food but not when food was supplied; thus, there were also no chronic effects of PET MPs. In contrast, intracellular reactive oxygen species levels and glutathione S-transferase activity in rotifers were significantly increased by PET MPs. Transcriptomic and metabolomic analyses revealed that genes and metabolites related to energy metabolism and immune processes were significantly affected by PET MPs in a concentration-dependent manner. Although acute toxicity of PET MPs was not observed, PET MPs are potentially toxic to the antioxidant system, immune system, and energy metabolism in rotifers.

Transcriptome and computational approaches highlighting the molecular regulation of Zacco platypus induced by mesocosm exposure to common disinfectant chlorine.

Chlorine (Cl2) is a disinfectant often used in swimming pools and water treatment facilities. However, it is released into aquatic ecosystems, where it may harm non-targeted organisms. Here, we performed a mesocosm experiment exposing Zacco platypus (Z. platypus) to biocide Cl2 for 30 days (30 d) at two days' time points 15 days (15 d) and 30 d samples were collected. Here, Z. platypus was exposed to a sublethal concentration (0.1 mg/L) of Cl2, and comparative transcriptomics analyses were performed to determine their response mechanisms at the molecular level. According to RNA sequencing of the whole-body transcriptome, 860 and 1189 differentially expressed genes (DEGs) were identified from the 15 d and 30 d responses to Cl2, respectively. After enrichment analysis of GO (Gene Ontology) functions and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, identified DEGs were demonstrated to be associated with a variety of functions, including "ion binding and transmembrane transporters". Cl2 also induced oxidative stress in Z. platypus by increasing the levels of reactive oxygen species (ROS) while decreasing the catalase (CAT) content and the levels of solute carrier family 22 member 11 (slc22a11), Caspase-8 (casp-8), inducible nitric oxide synthase (NOS2), cytosolic phospholipase A2 gamma (PLA2G4). However, Z. platypus still allows recovery during stress suspension by increasing the expression levels of solute carrier family proteins. The GO and KEGG annotation results revealed that the expression of DEGs were related to the detoxification process, immune response, and antioxidant mechanism. Additionally, protein-protein interaction networks (PPI) and cytoHubba analyses identified sixteen hub genes and their interaction. These findings elucidate the regulation of various DEGs and signaling pathways in response to Cl2 exposure, which will improve our knowledge and laid foundation for further investigation of the toxicity of Cl2 to Z. platypus.

Application of jetventurimixer for developing low-energy-demand and highly efficient aeration process of wastewater treatment.

In biological wastewater treatment, the oxygen supply in an aeration tank is the most important factor for removing organic pollutants, but it takes a large amount of electricity to generate the oxygen supply required. The Jetventurimixer (JVM) is a device that applies Bernoulli's principle, and the difference in flow rate pressure through the impeller is generated by the rotational force. Due to this physical mechanism, this device can supply oxygen in the atmosphere to the bioreactor without additional power. In this study, the JVM-based aeration process was developed for more efficient water treatment that demands lower energy. Parameters were measured for validating the efficiency and lower power demands, including the oxygen mass transfer characteristics and power efficiency. The results indicated that all parameters related to the oxygen mass transfer characteristics were advanced in performance by more than 200 % compared to those of the conventional air diffuser. In the case of power efficiency, it was confirmed that performance was 153-176 % higher. Therefore, it was confirmed that the JVM provides high-efficiency and low-energy benefits to the aeration process and, based on these advantages, the developed system seems to require further studies and validation for application to the water treatment system.

Metabolomic analysis predicted changes in growth rate in Daphnia magna exposed to acetaminophen.

As urbanization and the global population increases, pollutants associated with municipal wastewater such as pharmaceuticals are becoming more prevalent in aquatic environments. Acetaminophen (paracetamol) is a widely used drug worldwide and one of the most frequently detected pharmaceuticals in freshwater ecosystems. This study investigated the impact of acetaminophen on the metabolite profile of Daphnia magna at two life stages; and used these metabolomic findings to hypothesize a potential impact at a higher organismal level which was subsequently tested experimentally. Targeted polar metabolite analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to measure changes in the concentration of 51 metabolites in the neonate (> 24 h old) and adult (8 day-old) daphnids following a 48-h exposure to sub-lethal concentrations of acetaminophen. The impact of acetaminophen on the metabolic profile of neonates was widely different from adults. Also, acetaminophen exposure perturbed the abundance of nucleotides more extensively than other metabolites. The acute metabolomic experimental results led to the hypotheses that exposure to sub-lethal concentrations of acetaminophen upregulates protein synthesis in D. magna and subsequently increases growth during early life stages and has an opposite impact on adults. Accordingly, a 10 day growth rate experiment indicated that exposure to acetaminophen elevated biomass production in neonates but not in adults. These novel findings demonstrate that a targeted analysis and interpretation of the changes in the polar metabolic profile of organisms in response to environmental stressors could be used as a tool to predict changes at higher biological levels. As such, this study further emphasizes the incorporation of molecular-level platforms as critical and robust tools in environmental assessment frameworks and biomonitoring of aquatic ecosystems.

Pyrolytic Remediation and Ecotoxicity Assessment of Fuel-Oil-Contaminated Soil.

Oil-contaminated soil is a major societal problem for humans and the environment. In this study, the pyrolysis method was applied to oil-contaminated soil used as a landfill and gas station site in Korea. The removal efficiency of the main components of oil-contaminated soils, such as total petroleum hydrocarbons (TPH), polyaromatic hydrocarbons (PAHs), unresolved complex mixture (UCM), and alkylated PAHs (Alk-PAHs) were measured, and the effect of temperature, treatment time, and moisture content on pyrolysis efficiency was studied. In order to evaluate the risk of soil from which pollutants were removed through pyrolysis, integrated ecotoxicity was evaluated using Daphnia magna and Allivibrio fischeri. The chemical and biological measurements in this study include contaminants of emerging concerns (CECs). Results showed that the pyrolysis was more efficient with higher treatment temperatures, moisture content, and treatment times. In addition, toxicity was reduced by 99% after pyrolysis, and the degree of toxicity was evaluated more sensitively in Allivibrio fischeri than in Daphnia magna. This study shows that weathered oil-contaminated soil can be effectively treated in a relatively short time through pyrolysis, as well as provides information on efficient conditions and the assessment of ecotoxicity.

Comparative Study of the Synergistic Effects of Blending Raw/Torrefied Biomass and Vietnamese Anthracite Using Co-pyrolysis.

Biomass can be upgraded via torrefaction, and torrefied kenaf (TK) is a fuel that allows blending with coal at high ratios. In the present study, raw kenaf (Hibiscus cannabinus L.) (RK) was torrefied at 523 K for 30 min and then mixed with Vietnamese anthracite (NinhBinh, NB) before co-pyrolysis. Thermogravimetric (TG) analysis was used to evaluate the behavior of RK, TK, and blended RK/TK during co-pyrolysis at biomass blending ratios (BBRs) of 0, 25, 50, 75, and 100 wt %. The TG and derivative thermogravimetry curves of a mixture of NB and RK (NBRK) were similar to those of RK. The decomposition curves of a mixture of NB and TK (NBTK) depended on the mass fraction of TK. Based on weight loss differences between the experimental and calculated data for the fuel blends, no interaction between the RK and anthracite was observed for all BBRs, whereas anthracite involving 50 and 75% TK exhibited synergistic effects. The temperature range for synergy and degree of synergy for NB and TK depended on the heating rate and mass ratio of TK. Kinetic parameters were calculated using the Friedman-non-isothermal free kinetic method at heating rates of 10, 20, and 40 K/min. The results showed that the activation energy (E) values of the NBRK at conversion ratios of 0.2-0.5 were equal to those of the RK, whereas they were superior at NB decomposition ratios of 0.6-0.8. NBTK1-1 (BBR of 50%) showed E values higher than those of NB at some conversion ratios, thus demonstrating a negative impact of blending. Further, NBTK1-3 (BBR of 75%) and NBTK3-1 (BBR of 25%) exhibited E values between those of NB and TK. The present study suggests that a high TK mass fraction (75%) in the blend for co-pyrolysis is optimal for the activation energy and volatile matter yield.

Antioxidant enzyme activity in Daphnia magna under microscopic observation and shed carapace length as an alternative growth endpoint.

Daphnia magna is an important organism for exposure studies in ecotoxicology. Body length measurement of a daphnid is a useful endpoint that represents the adverse effects of exposed chemicals or conditions on growth. This study questioned whether stress on body length measurement conditions while maintaining Daphnia magna on a slide glass results in any impact to antioxidant enzyme activity or growth. At the same time, the shed carapace length was tested to see if it could predict body length and be used as an alternative endpoint. The measured catalase (CAT) activity decreased as the exposure time to the on-slide conditions increased from 30 s to 120 s, although glutathione-S-transferase (GST) activity was not affected. On the other hand, regression between body length and two parameters of shed carapace length showed R2 values, 0.869 and 0.924. Growth measured for each molting for three weeks was not affected by the exposure to the on-slide conditions. Finally, this study confirmed potential oxidative stress based on the exposure time dependent CAT activity in Daphnia magna under microscopic observation. More importantly, the shed carapace length was validated to reflect body length and it implies applicability of the new parameters to sublethal effect measurement using Daphnia magna. This study suggests potential interference is possible with the traditional growth measurement method on antioxidant enzyme activity in Daphnia magna and proposes better experimental practices to avoid the interference.