Influence of Ultrafine Bubbles (UFBs) on Cadmium Toxicity in <i>Polypedilum nubifer</i> (Chironomidae)

Kabul Fadilah; Yosuke Sawada; Kenji Kamura; Takeshi Fujino

doi:10.5620/eaht.2026010

Environ Anal Health Toxicol > Volume 41:2026 > Article

Fadilah, Sawada, Kamura, and Fujino: Influence of Ultrafine Bubbles (UFBs) on Cadmium Toxicity in Polypedilum nubifer (Chironomidae)

Original Article

Environ Anal Health Toxicol 2026; 41(1): e2026010.

Published online: March 26, 2026

DOI: https://doi.org/10.5620/eaht.2026010

Influence of Ultrafine Bubbles (UFBs) on Cadmium Toxicity in Polypedilum nubifer (Chironomidae)

Kabul Fadilah^1,^2,^*

, Yosuke Sawada³, Kenji Kamura³, Takeshi Fujino¹

¹Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-Ku, Saitama 338-8570, Japan

²Department of Environmental Engineering, Faculty of Engineering, Universitas Pembangunan Nasional Veteran Jawa Timur, Surabaya, 60294 Indonesia

³MARUYAMA MFG. CO., INC., Tokyo 101-0047, Japan

^*Correspondence: kabul.fadilah.tl@upnjatim.ac.id, fujino@mail.saitama-u.ac.jp

Recommended by: Prof. Ha Ryong Kim

Received December 23, 2025 Accepted March 3, 2026

This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Cadmium is one of the major pollutants that threatens aquatic ecosystems. Aquatic organisms such as aquatic insects are commonly used as bioindicators to assess the impact of pollutants in the environment. Recent studies have demonstrated that ultrafine bubbles (UFBs), gas-filled nanobubbles with unique properties, can modulate metal toxicity in aquatic organisms. However, their role in toxicity assessments involving aquatic insects remains limited. In this study, the effects of UFBs on Cd-induced toxicity in Polypedilum nubifer were investigated. Cadmium exposure led to inhibited larval growth, reduced adult emergence, increased activity of biochemical markers, and greater Cd accumulation in larval tissues. When co-treated with UFBs, survival, larval growth and adult emergence did not differ significantly compared to the Cd treatment alone. However, UFB treatment did lead to a significant reduction in internal Cd accumulation. Furthermore, the activities of catalase (CAT) and glutathione S-transferase (GST), which increased under Cd exposure, were reduced to levels that aligned closely with the control group. In conclusion, this study provides the potential application of UFB for mitigating heavy metal pollution at a physiological level, specifically by lowering heavy metal accumulation and modulating oxidative stress–related defense responses.

Keywords: Cadmium, ultrafine bubbles, Polypedilum nubifer, accumulation; biomarker

Introduction

Heavy metal pollution, particularly cadmium (Cd), poses significant threats to aquatic ecosystems and their presence come from such as industrial discharge, mining operations, agricultural runoff, and domestic waste [1,2]. When introduced into aquatic environments, Cd readily accumulates in sediments and biota, posing substantial risks to both ecosystem health through biomagnification and food web transfer [3,4]. Due to its high toxicity, persistence, and capacity for biological accumulation [5,6] Cd could disrupt metabolic processes in aquatic organisms.

Aquatic insects such as chironomids are recognized as sensitive bioindicators of environmental pollution. Moreover, it is easy to maintain under laboratory conditions and exhibits high reproductive capacity. They have complex life cycle, which experiences a complete metamorphosis involving larval, pupal, and adult stages [7]. In chironomids toxicity test, frequently measured endpoints comprise survival, growth, and development as observable effects for early indicators of environmental stressors [8]. Polypedilum nubifer, a freshwater midge belonging to the Chironomidae family, is common species of midge found in aquatic habitats across Japan and serves as a representative model for assessing pollutant toxicity [9].

Ultrafine bubbles (UFBs) defined as gas bubbles with diameters smaller than 1 μm with unique properties such as high stability, high zeta potential and large surface area. These characteristics have been exploited in various applications. In aquaculture, for example, their use has been shown to improve the production rate of striped catfish [10]. They are also known to enhance seed germination and growth of plants for agriculture purposes [11,12]. In water treatment, UFBs application have demonstrated potential effect for enhancing the removal of contaminants through several mechanisms such as improved gas dissolution, surface adsorption, and the generation of reactive oxygen species (ROS) upon bubble collapse [13–15]. These properties make UFBs a valuable approach for mitigating pollutants in aquatic environments. Nevertheless, the potential role of UFBs in pollution mitigation and in supporting aquatic life has not been widely explored.

Recently, UFBs have been studied for toxicity assessment. UFBs application showed influence on the toxicity and physiological responses of several organism, such as Daphnia magna, Chlorella vulgaris and Oryza sativa, including the activity of antioxidant enzymes [16–18]. However, the effects of UFBs on organisms with more complex life stages, such as chironomids, remain unknown. Considering the emerging potential of UFBs in reducing pollutant toxicity, investigating their interaction offers a promising direction for ecotoxicological research.

Therefore, this study aimed to evaluate the effects of UFBs on Cd exposure in P. nubifer. The survival, growth parameters, Cd accumulation, and biochemical biomarkers such as catalase (CAT) and glutathione S-transferase (GST) were measured. The results of this research will provide new insights into the effects of UFBs on the interaction between metal toxicity and aquatic organisms, as well as a basis for supporting sustainable environmental applications.

Materials and Methods

Test Organism

P. nubifer were obtained from cultured containers in the Applied Ecological Engineering Laboratory, Saitama University. Larvae were reared in plastic containers (27.7 x 20.5 x 4.3 cm) at 20±2 °C under a 16:8 light: dark cycle with a 1 cm layer of GEX Aqua natural sand sediment (<1 mm). Commercial fish food (TetraMin®, Tetrawerke, Melle, Germany) was used to feed the larvae three times a week.

Ultrafine Bubble Preparation

UFB water was generated by circulating dechlorinated tap water through the UFB generator (UP0290M-1 1.4 L/min, 2 MPa, 250 W) MARUYAMA MFG. CO., INC., Tokyo, Japan. The size of UFBs and zeta potential were measured by ELSZ-2000 (Otsuka Electronics, Hirakata, Japan). In addition, water quality parameters were also measured. Dissolved concentrations (DO) values were determined using HQ30d (Hach, USA), and pH values were measured using a pH meter (Mettler Toledo, Switzerland). The data are presented in Table S1.

Experimental Design

A 1000 mg/L CdCl₂ stock solution (Fujifilm, Wako, Japan) was prepared by diluted in distilled water. This experiment consists of four groups: (1) Control (tap water), (2) UFB only, (3) Cd and (4) UFB-Cd. Each experiment was conducted in 250 mL glass bottles containing 100 mL of test water and a 1 cm layer of natural sand sediment, containing 10 organisms per replicate. The experiment was maintained at 20±1 °C and a 16:8-h light:dark photoperiod.

Survival, Growth and Adult Emergence

First instar P. nubifer larvae were exposed to four different groups for a period of 10 days. In this experiment, 50 μg/L Cd was used for toxicity assessment. This concentration was selected after the preliminary experiment using 12.5, 25, 50 and 100 μg/L, which identified 50 μg/L as the highest concentration producing measurable biological responses without causing excessive mortality. The exposure medium was renewed every two days. Survival and growth parameters (body length) of P. nubifer larvae were determined on day 10 from the first three replicates. Survived larvae were collected, and body length measured under stereo-microscope (Olympus SZ61, Tokyo, Japan). The three remaining replicates were monitored for adult emergence and were recorded daily for 28 days. Measurements of water physicochemical parameters were carried out throughout the test (Table S2)

Cadmium Accumulation in P. nubifer Larvae

Fourth instar larvae were exposed to 500 μg/L Cd for 48 h. A higher Cd concentration was applied to ensure sufficient internal Cd accumulation for reliable quantification above analytical detection limits and to allow assessment of UFB effects on metal bioaccumulation. After exposure, larvae were washed with phosphate buffer to remove Cd from the larvae body and dried at 65 °C for 24 h. Larvae were acid wet digested in a mixture of 3 mL HNO₃ at 125 ºC for 1 h. Subsequently, 1 mL of H₂O₂ was added to the mixture to oxidize the organic matter completely. After acid wet digestion, the samples were adjusted to 10 mL with distilled water. The Cd concentration was determined using an Atomic Absorption Spectrophotometer (AA7000, Shimadzu, Japan) with a detection limit for Cd of 0.01 mg/L.

Biochemical Marker Analysis

For biochemical marker analysis, fourth instar larvae of P. nubifer were exposed to 500 μg/L Cd for 24 h without feeding. This concentration was selected to induce detectable oxidative stress responses within a short exposure period while avoiding severe lethality, allowing reliable measurement of enzymatic activity. At the end of the exposure, larvae were removed from each treatment and homogenized with potassium phosphate (pH 7.4, 100 mM) and centrifuged at 10000 × g for 10 min at 4 °C. The supernatants were collected and used for evaluation of catalase (CAT) and glutathione S-transferase (GST) activity.

Catalase activity was determined on 50 μg/L of the homogenate in the mixture of 100 mM potassium phosphate buffer (pH 7.0) and 20.0 mM hydrogen peroxide (H₂O₂) in a 1:1 (v/v) ratio following a protocol adapted from [19]. Catalase activity was monitored by decreasing H₂O₂ absorbance at 240 nm for 3 minutes and expressed in units of μmol/min/mg protein.

Glutathione S-transferase activity was measured following the method of Habig and Jakoby [20]. In 1 mL microtubes, 50.0 μL of homogenate mixed with a solution of 100 mM potassium phosphate buffer (pH 6.5), 1.0 mM 1-chloro-2, 4-dinitrobenzene (CDNB) and 10 mM reduced glutathione (GSH). The activity was measured at a 340 nm wavelength for 5 minutes and quantified in units of nmol/min/mg protein. Protein concentration was measured according to BCA Protein Assay Kit (Pierce™, Thermo Fisher Scientific) using bovine serum albumin (BSA) as a standard.

Data Analysis

Data were tested for normality and homogeneity of variances using the Shapiro–Wilk and Brown-Forsythe tests, respectively. One-way analysis of variance (ANOVA) was applied, followed by Tukey’s post hoc test to identify significant differences among treatment groups. The non-parametric Kruskal-Wallis test was used to analyze the survival of P. nubifer. Differences between groups were considered significant when p < 0.05. All statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software Inc., USA).

Results

Ultrfine Bubbles Properties

The UFB water in this experiment showed distinct physical and chemical properties, with a particle size of 244.33±39.9 nm and a zeta potential of –9.1±0.79 mV. In contrast, the control water showed no detectable particle size and zeta potential of 0.48±0.24 mV. The small particle size indicates a high surface-area-to-volume ratio, potentially influencing interactions with dissolved substances and biological membranes [21]. The negative zeta potential of UFB water indicates that the bubbles possess negatively charged surfaces, which contributes to their stability in solution through electrostatic repulsion that prevents bubble coalescence and collapse [22].

Survival of P. nubifer

Survival assessment after 10 days of exposure showed high tolerance of P. nubifer to Cd exposure, but the effects were not significant differences among treatments (Fig. 1, p > 0.05). The control and UFB-only groups presented a survival rate of 100%. Meanwhile both the Cd and UFB-Cd groups showed survival rates of 96.67%.

Growth of P. nubifer

Evaluating larval growth through body length measurements provided an environmentally relevant and detailed assessment of Cd effects. As shown in Fig. 2, the body length was significantly influenced by experimental treatment (p < 0.05). The control group had similar growth to those exposed solely to UFB water (p > 0.05). Meanwhile, the influence of Cd exposure reduced the body length in the Cd and UFB-Cd groups, but not significantly different (p > 0.05), approximately 23% and 8% reduction compared to the control, respectively.

Adult Emergence of P. nubifer

Adult emergence rates provided additional insights into the long-term effects and ecological indicator of Cd toxicity. In this study, control groups showed an emergence rate of 83.33%, while UFB-treated groups exhibited a slightly lower but comparable rate of 80%. Meanwhile, the Cd group showed reduced adult emergence to 20%, indicating severe impacts of Cd exposure that disrupt metamorphosis processes. Compared to the Cd group, the UFB-Cd treatment did not differ significantly from each other (p > 0.05). In addition, the timing of adult emergence was also delayed in both Cd and UFB-Cd groups compared to the control and UFB. In the Control and UFB treatments, emergence began on day 15, while in the Cd-exposed groups, it was delayed until day 19 (Fig. 3).

Cadmium Accumulation in P. nubifer

Cadmium accumulation in P. nubifer larvae following 48-hour revealed significant differences between treatment groups (p < 0.05). In the Cd group (Fig. 4), larvae exposed to 500 μg/L accumulated the highest levels of Cd (304.45 μg/g dw). In contrast, larvae at UFB-Cd treatment accumulated Cd concentration (224.81 μg/g dw), which was significantly lower compared to Cd group (p < 0.05). Meanwhile Control and UFB group showed no Cd accumulation.

Biochemical Marker Analysis

To assess the cellular level effects and early biochemical alterations associated with Cd exposure in P. nubifer larvae, the activities of two biomarkers, catalase (CAT) and glutathione S-transferase (GST), were examined. In the present study (Fig. 5), similar trends were observed for CAT and GST activity. Compared to other treatments, CAT (Fig. 5a, p < 0.05) and GST (Fig. 5b, p < 0.05) activities significantly increased in the Cd group, indicating activation of antioxidant and detoxification pathways in response to Cd-induced cellular challenge. Interestingly, the UFB-Cd group showed significantly reduced enzyme activities compared to the Cd group (p < 0.05), but did not differ significantly from control and UFB group (p > 0.05) with values close to those observed in the control group.

Discussion

In general, Cd exposure disrupts the regulation of internal ion stability and triggers oxidative stress through the production of reactive oxygen species (ROS) in aquatic organisms, which may contribute to decreased survival [23,24]. In this case, despite a small reduction in survival was observed, the result indicates the ability of P. nubifer larvae to tolerate Cd exposure. This finding aligns with previous studies reported by Sildanchandra and Crane [28], which demonstrated that Chironomus riparius maintained 100% survival after 10 days of exposure to Cd at 250 μg/L. This tolerance is likely due to the larvae's physiological mechanisms, as reported by Griffith [26] in which the aquatic insects maintain ionic equilibrium and facilitate Cd detoxification. However, this tolerance level may be overwhelmed under extreme exposure levels. Nevertheless, long-term exposure at higher doses can compromise physiological processes, leading to metabolic disruption and cellular damage [27]. Although UFB treatment in this study did not significantly enhance survival rate, its influence might be more relevant at higher concentrations, potentially through reducing Cd toxicity or modulating stress responses [18,28].

The reduced growth in larvae may be explained by disruptions in metabolic and physiological processes including impaired protein synthesis, hormonal balance disturbances, and compromised energy metabolism pathways [29–31]. At the molecular level, Cd exposure stimulates the production of metallothioneins [32], proteins that bind Cd ions to reduce toxicity. However, under prolonged exposure, this defense mechanism may become saturated, leading to cellular dysfunction and ultimately inhibiting the growth [33]. Several studies have demonstrated that Cd exposure can impair organismal growth, as shown by significant growth inhibition and reduced feed efficiency in rainbow trout [34], altered glucose metabolism and cellular stress in juvenile channel catfish under chronic exposure [35], and disrupted embryonic development in Daphnia magna [36]. The reduced adult and time emergence may be attributed to the disruption during developmental processes, which need more energy allocation due to toxic exposure [37]. Consequently, it impaired growth, as observed in this result with reduced body length of P. nubifer larvae. Furthermore, Cd interferes with metamorphosis by disrupting ecdysone hormone regulation [38,39], which is essential for successful molting and developmental transitions.

To date, research examining the influence of UFB application on the growth of aquatic insects or other aquatic animals under heavy metal exposure remains limited. In the present study, despite reduced Cd accumulation and moderated biochemical responses, UFB exposure did not significantly improve larval growth and adult emergence under the chronic Cd concentration tested. Prolonged Cd exposure at 50 μg/L may impose a level of chronic stress exceeding the capacity of UFB-mediated modulation to promote growth recovery. In addition, residual Cd levels within larval tissues may have continued to interfere with developmental processes, thereby constraining recovery at the organism level. This indicate that that while UFB influenced Cd bioavailability and physiological processes, these changes were insufficient to translate into measurable improvements in growth. Organism-level endpoints such as growth and emergence are cumulative and integrate long-term energetic and developmental costs, and therefore may not respond proportionally to partial reductions in metal uptake or biochemical changes alone. Individual biomarker responses have limited predictive power for whole-organism effects because they reflect mechanistic changes at lower levels of biological organization rather than integrated life-history outcomes [40]. Nevertheless, this interpretation is constrained by the specific exposure concentration and experimental design employed. Thus, additional studies are needed to clarify the effects of UFB treatment under varying Cd concentrations and exposure scenarios in P. nubifer larvae and other aquatic insects.

Cadmium uptake in P. nubifer larvae occurs mainly through calcium transport pathways, as Cd competes with Ca²⁺ for shared uptake sites across gut and excretory epithelia. After uptake, Cd accumulates in ionoregulatory tissues such as the midgut and malpighian tubules [27,41]. The observed reduction in Cd accumulation under UFB treatment may be attributed to reduced bioavailability, possibly due to the physical and chemical properties of UFBs. UFBs possess a highly negative zeta potential due to the preferential adsorption of hydroxide ions at the gas–liquid interface [14,42], creating a charged surface that can attract and temporarily bind divalent metal cations such as Cd²⁺ [43]. This interaction could reduce freely dissolved Cd²⁺ ions available for biological uptake, as similarly suggested in hydroponic and aquatic plant studies [18,44]. However, in the present study, direct measurements of Cd speciation or adsorption onto UFBs were not conducted. Future studies should quantify Cd partitioning between dissolved and UFB-associated fractions and examine UFB stability and surface charge dynamics under exposure conditions to clarify the specific pathways by which UFBs influence metal bioavailability.

The increased levels of CAT and GST enzymes in the Cd group, represent a defensive response against Cd-induced oxidative stress in P. nubifer larvae. Catalase plays a pivotal role in cellular defense mechanisms to prevent the formation of highly reactive hydroxyl radicals by converting hydrogen peroxide into H₂O and O₂ [45]. Glutathione S-transferase (GST) is a vital phase II detoxification enzyme that conjugates reactive electrophilic compounds with glutathione. Simultaneously, GST contributes to the neutralization and excretion of heavy metals such as Cd [46]. This result is supported by earlier studies that demonstrated elevated antioxidant enzyme activities in aquatic organisms following heavy metal exposure, particularly at low level concentrations [47–49].

The presence of UFBs appeared to influence Cd-associated biochemical responses in P. nubifer larvae, as indicated by lower CAT and GST activities compared to the Cd-only group. This reduction may reflect a decrease in oxidative challenge, potentially resulting from reduced Cd uptake and lower intracellular Cd²⁺ concentrations. In addition, negative charged of UFB may improve the physiological status of P. nubifer larvae by enhancing the metabolic activity [50], thus enabling the larvae to adapt and increase detoxification capacity against Cd exposure. A similar results were also found by Fan et al. [16] and Liu et al. [17] which reported that the application of hydrogen nanobubbles decreased reactive oxygen species (ROS) levels in Daphnia magna and Chlorella vulgaris under copper stress. Although these enzymes provide useful information on antioxidant and detoxification-related cellular responses, they do not capture the full spectrum of oxidative stress processes or oxidative damage. Additional endpoints, such as reactive oxygen species production, lipid peroxidation, or other antioxidant enzymes, would be necessary to comprehensively assess redox imbalance and oxidative injury.

Conclusions

The result of this research demonstrated that UFBs significantly reduced the accumulation of Cd in larval tissues and moderated Cd-induced biochemical responses in P. nubifer larvae. While larval survival, growth and adult emergence showed no significant improvement with UFB exposure. These findings suggest that while UFBs can modulate Cd bioavailability and associated cellular defense responses, these effects did not yield a significant improvement of larval growth and adult emergence under the experiment conditions. Future study is required to investigate the mechanism of metal uptake and interaction with UFBs during the exposure to provide a better understanding of the effects of toxicity to the organism.

Notes

Acknowledgement

This work was supported by MARUYAMA MFG. CO., INC., Tokyo, Japan. We would like to thank Srijana Thapa Magar and Zhang Yi for their support during the experiment.

Conflict of interest

The authors declare no conflicts of interest.

CRediT author statement

KF: Conceptualization, Methodology, Formal Analysis, Writing - Original draft Preparation. YS: Conceptualization, Methodology. KK: Conceptualization, Methodology. TF: Conceptualization, Methodology, Writing - Review & Editing, Supervision.

Supplementary Material

Add short descriptions of supplementary material. This material is available online at www.eaht.org.

eaht-41-1-e2026010-Supplementary-Table-S1-S4.pdf

References

1. Zhao XM, Yao LA, Ma QL, Zhou GJ, Wang L, Fang QL, et al. Distribution and ecological risk assessment of cadmium in water and sediment in Longjiang River, China: implication on water quality management after pollution accident. Chemosphere 2018;194: 107-116 https://doi.org/10.1016/j.chemosphere.2017.11.127.

2. Yuan Z, Luo T, Liu X, Hua H, Zhuang Y, Zhang X, et al. Tracing anthropogenic cadmium emissions: from sources to pollution. Sci Total Environ 2019;676: 87-96 https://doi.org/10.1016/j.scitotenv.2019.04.250.

3. Suhani I, Sahab S, Srivastava V, Singh RP. Impact of cadmium pollution on food safety and human health. Curr Opin Toxicol 2021;27: 1-7 https://doi.org/10.1016/j.cotox.2021.04.004.

4. Luo W, Zhang Y, Zhang S, Sun K, Li K, He F, et al. Cadmium accumulation characteristics and dietary risks in three aquatic animals with different habitat characteristics in a rice-fish co-culture system. Aquac Rep 2024;39: 102417 https://doi.org/10.1016/j.aqrep.2024.102417.

5. Zhang H, Reynolds M. Cadmium exposure in living organisms: a short review. Sci Total Environ 2019;678: 761-767 https://doi.org/10.1016/j.scitotenv.2019.04.395.

6. Saravanan P, Saravanan V, Rajeshkannan R, Arnica G, Rajasimman M, Baskar G, et al. Comprehensive review on toxic heavy metals in the aquatic system: sources, identification, treatment strategies, and health risk assessment. Environ Res 2024;258: 119440 https://doi.org/10.1016/j.envres.2024.119440.

7. Czechowski P, Stevens MI, Madden C, Weinstein P. Steps towards a more efficient use of chironomids as bioindicators for freshwater bioassessment: exploiting eDNA and other genetic tools. Ecol Indic 2020;110: 105868 https://doi.org/10.1016/j.ecolind.2019.105868.

8. Vogt C, Belz D, Galluba S, Nowak C, Oetken M, Oehlmann J. Effects of cadmium and tributyltin on development and reproduction of the non-biting midge Chironomus riparius (Diptera)—baseline experiments for future multi-generation studies. J Environ Sci Health A Tox Hazard Subst Environ Eng 2007;42(1):1-9 https://doi.org/10.1080/10934520601015255.

9. Hatakeyama S. Chronic effects of Cd on reproduction of Polypedilum nubifer (Chironomidae) through water and food. Environ Pollut 1987;48(4):249-261 https://doi.org/10.1016/0269-7491(87)90106-0.

10. Subhan U, Panatarani C, Joni IM. Effect of ultrafine bubbles on various stocking density of striped catfish larviculture in recirculating aquaculture system. Fishes 2022;7(4):190 https://doi.org/10.3390/fishes7040190.

11. Ahmed AKA, Shi X, Hua L, Manzueta L, Qing W, Marhaba T, et al. Influences of air, oxygen, nitrogen, and carbon dioxide nanobubbles on seed germination and plant growth. J Agric Food Chem 2018;66(20):5117-5124 https://doi.org/10.1021/acs.jafc.8b00333.

12. Wang Y, Wang S, Sun J, Dai H, Zhang B, Xiang W, et al. Nanobubbles promote nutrient utilization and plant growth in rice by upregulating nutrient uptake genes and stimulating growth hormone production. Sci Total Environ 2021;800: 149627 https://doi.org/10.1016/j.scitotenv.2021.149627.

13. Agarwal A, Ng WJ, Liu Y. Principle and applications of microbubble and nanobubble technology for water treatment. Chemosphere 2011;84(9):1175-1180 https://doi.org/10.1016/j.chemosphere.2011.05.054.

14. Wu J, Zhang K, Cen C, Wu X, Mao R, Zheng Y. Role of bulk nanobubbles in removing organic pollutants in wastewater treatment. AMB Express 2021;11: 96 https://doi.org/10.1186/s13568-021-01254-0.

15. Zhou S, Qiao L, Jia Y, Khanal SK, Sun L, Lu H. Micro-nano bubble ozonation for effective treatment of ibuprofen-laden wastewater and enhanced anaerobic digestion performance. Water Res 2025;273: 123006 https://doi.org/10.1016/j.watres.2024.123006.

16. Fan W, Zhang Y, Liu S, Li X, Li J. Alleviation of copper toxicity in Daphnia magna by hydrogen nanobubble water. J Hazard Mater 2020;389: 122155 https://doi.org/10.1016/j.jhazmat.2020.122155.

17. Liu S, Li J, Oshita S, Kamruzzaman M, Cui M, Fan W. Formation of a hydrogen radical in hydrogen nanobubble water and its effect on copper toxicity in Chlorella. ACS Sustain Chem Eng 2021;9(33):11100-11109 https://doi.org/10.1021/acssuschemeng.1c02936.

18. Huang M, Nhung NTH, Wu Y, He C, Wang K, Yang S, et al. Different nanobubbles mitigate cadmium toxicity and accumulation of rice (Oryza sativa L.) seedlings in hydroponic cultures. Chemosphere 2023;312: 137250 https://doi.org/10.1016/j.chemosphere.2022.137250.

19. Claiborne A. Catalase activity. Handb Methods Oxyg Radic Res. 1985. p. 283-284.

20. Habig WH, Jakoby WB. Assays for differentiation of glutathione S-transferases. Methods Enzymol 1981;77: 398-405 https://doi.org/10.1016/S0076-6879(81)77053-8.

21. Hansen HH, Cha H, Ouyang L, Zhang J, Jin B, Stratton H, et al. Nanobubble technologies: applications in therapy from molecular to cellular level. Biotechnol Adv 2023;63: 108091 https://doi.org/10.1016/j.biotechadv.2022.108091.

22. Ushikubo FY, Furukawa T, Nakagawa R, Enari M, Makino Y, Kawagoe Y, et al. Evidence of the existence and the stability of nano-bubbles in water. Colloids Surf A Physicochem Eng Asp 2010;361(1-3):31-37 https://doi.org/10.1016/j.colsurfa.2010.03.005.

23. Chen X, Li H, Zhang J, Ding Y, You J. Does cadmium affect the toxicokinetics of permethrin in Chironomus dilutus at sublethal level? Evidence of enzymatic activity and gene expression. Environ Pollut 2016;218: 1005-1013 https://doi.org/10.1016/j.envpol.2016.08.051.

24. Rangaswamy B, Kim WS, Kong D, Kwak IS. Transcriptomic traces of adaptive changes and detoxification in Glyptotendipes tokunagai (Chironomidae) exposed to cadmium-based sediments environment. Environ Adv 2024;16: 100523 https://doi.org/10.1016/j.envadv.2024.100523.

25. Sildanchandra W, Crane M. Influence of sexual dimorphism in Chironomus riparius Meigen on toxic effects of cadmium. Environ Toxicol Chem 2000;19(9):2309-2313 https://doi.org/10.1002/etc.5620190921.

26. Griffith MB. Toxicological perspective on the osmoregulation and ionoregulation physiology of major ions by freshwater animals: teleost fish, crustacea, aquatic insects, and mollusca. Environ Toxicol Chem 2017;36(3):576-600 https://doi.org/10.1002/etc.3676.

27. Gillis PL, Wood CM. The effect of extreme waterborne cadmium exposure on the internal concentrations of cadmium, calcium, and sodium in Chironomus riparius larvae. Ecotoxicol Environ Saf 2008;71(1):56-64 https://doi.org/10.1016/j.ecoenv.2007.08.003.

28. Yan D, Xue S, Zhang Z, Xu G, Zhang Y, Gao J, et al. Air nanobubble water improves plant uptake and tolerance toward cadmium in phytoremediation. Environ Pollut 2023;337: 122577 https://doi.org/10.1016/j.envpol.2023.122577.

29. Choi J, Ha MH. Effect of cadmium exposure on the globin protein expression in 4th instar larvae of Chironomus riparius Mg. (Diptera: Chironomidae): an ecotoxicoproteomics approach. Proteomics 2009;9(1):31-39 https://doi.org/10.1002/pmic.200701197.

30. Sokolova IM, Frederich M, Bagwe R, Lannig G, Sukhotin AA. Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates. Mar Environ Res 2012;79: 1-15 https://doi.org/10.1016/j.marenvres.2012.04.003.

31. Sandhu N, McGeer JC, Vijayan MM. Exposure to environmental levels of waterborne cadmium impacts corticosteroidogenic and metabolic capacities, and compromises secondary stressor performance in rainbow trout. Aquat Toxicol 2014;146: 20-27 https://doi.org/10.1016/j.aquatox.2013.10.010.

32. Gillis PL, Diener LC, Reynoldson TB, Dixon DG. Cadmium-induced production of a metallothioneinlike protein in Tubifex tubifex (Oligochaeta) and Chironomus riparius (Diptera): correlation with reproduction and growth. Environ Toxicol Chem 2002;21(9):1836-1844 https://doi.org/10.1002/etc.5620210911.

33. Yan L, Sun C, Sun L, Cao C. Role of metallothionein gene in Cd and Pb detoxification in Chironomus kiiensis. Ecotoxicology 2025;34(5):666-676 https://doi.org/10.1007/s10646-025-02865-0.

34. Heydarnejad MS, Khosravian-Hemamai M, Nematollahi A. Effects of cadmium at sub-lethal concentration on growth and biochemical parameters in rainbow trout (Oncorhynchus mykiss). Ir Vet J 2013;66: 11 https://doi.org/10.1186/2046-0481-66-11.

35. Paul JS, Small BC. Chronic exposure to environmental cadmium affects growth and survival, cellular stress, and glucose metabolism in juvenile channel catfish (Ictalurus punctatus). Aquat Toxicol 2021;230: 105705 https://doi.org/10.1016/j.aquatox.2020.105705.

36. Pérez E, Hoang TC. Responses of Daphnia magna to chronic exposure of cadmium and nickel mixtures. Chemosphere 2018;208: 991-1001 https://doi.org/10.1016/j.chemosphere.2018.06.063.

37. Humann-Guilleminot S, Fuentes A, Maria A, Couzi P, Siaussat D. Cadmium and phthalate impacts developmental growth and mortality of Spodoptera littoralis, but not reproductive success. Ecotoxicol Environ Saf 2024;281: 116605 https://doi.org/10.1016/j.ecoenv.2024.116605.

38. Planelló R, Martínez-Guitarte JL, Morcillo G. Effect of acute exposure to cadmium on the expression of heat-shock and hormone-nuclear receptor genes in the aquatic midge Chironomus riparius. Sci Total Environ 2010;408(7):1598-1603 https://doi.org/10.1016/j.scitotenv.2010.01.004.

39. Rodríguez EM, Medesani DA, Fingerman M. Endocrine disruption in crustaceans due to pollutants: a review. Comp Biochem Physiol A Mol Integr Physiol 2007;146(4):661-671 https://doi.org/10.1016/j.cbpa.2006.04.030.

40. Forbes VE, Palmqvist A, Bach L. The use and misuse of biomarkers in ecotoxicology. Environ Toxicol Chem 2006;25(1):272-280 https://doi.org/10.1897/05-257R.1.

41. Leonard EM, Belowitz R, Agema P, O’Donnell MJ. Characterization of cadmium and calcium fluxes along the gut, malpighian tubules, and anal papillae of the dipteran Chironomus riparius. Environ Toxicol Chem 2018;37(10):2542-2549 https://doi.org/10.1002/etc.4211.

42. Dockar D, Sullivan P, Mifsud J, Gibelli L, Borg MK. Ionic adsorption on bulk nanobubble interfaces and its uncertain role in diffusive stability. J Colloid Interface Sci 2025;695: 137747 https://doi.org/10.1016/j.jcis.2025.137747.

43. Hewage SA, Kewalramani J, Meegoda JN. Stability of nanobubbles in different salts solutions. Colloids Surf Physicochem Eng Asp 2021;609: 125669 https://doi.org/10.1016/j.colsurfa.2020.125669.

44. Guo X, Chen R, Li H, Li G, Zhao Q, Wang D, et al. Micro-nano bubble hydrogen water irrigation improves the Cd tolerance of Ipomoea aquatica Forssk.: rhizosphere micro-biotas modulation and oxidative stress alleviation. Environ Pollut 2025;376: 126394 https://doi.org/10.1016/j.envpol.2025.126394.

45. Wu H, Xuan R, Li Y, Zhang X, Jing W, Wang L. Biochemical, histological and ultrastructural alterations of the alimentary system in the freshwater crab Sinopotamon henanense subchronically exposed to cadmium. Ecotoxicology 2014;23(1):65-75 https://doi.org/10.1007/s10646-013-1152-z.

46. Nair PMG, Choi J. Identification, characterization and expression profiles of Chironomus riparius glutathione S-transferase (GST) genes in response to cadmium and silver nanoparticles exposure. Aquat Toxicol 2011;101(3-4):550-560 https://doi.org/10.1016/j.aquatox.2010.12.006.

47. Ensibi C, Daly Yahia MN. Toxicity assessment of cadmium chloride on planktonic copepods Centropages ponticus using biochemical markers. Toxicol Rep 2017;4: 83-88 https://doi.org/10.1016/j.toxrep.2017.01.005.

48. Won EJ, Kim RO, Rhee JS, Park GS, Lee J, Shin KH, et al. Response of glutathione S-transferase (GST) genes to cadmium exposure in the marine pollution indicator worm, Perinereis nuntia. Comp Biochem Physiol C Toxicol Pharmacol 2011;154(2):82-92 https://doi.org/10.1016/j.cbpc.2011.03.008.

49. Xia L, Chen S, Dahms HU, Ying X, Peng X. Cadmium induced oxidative damage and apoptosis in the hepatopancreas of Meretrix meretrix. Ecotoxicology 2016;25(5):959-969 https://doi.org/10.1007/s10646-016-1653-7.

50. Liu S, Kawagoe Y, Makino Y, Oshita S. Effects of nanobubbles on the physicochemical properties of water: the basis for peculiar properties of water containing nanobubbles. Chem Eng Sci 2013;93: 250-256 https://doi.org/10.1016/j.ces.2013.02.004.

Figure 1.

Survival of P. nubifer after 10 days of Cd exposure. The data presents mean values ± SD. Different letters denote significant differences (p < 0.05).

Figure 2.

Body length of P. nubifer larvae after 10 days of Cd exposure. The data presents mean values ± SD. Different letters denote significant differences (p < 0.05).

Figure 3.

Cumulative adult emergence (%) of P. nubifer. The data presents mean values ± SD. Different letters denote significant differences (p < 0.05).

Figure 4.

Accumulation of Cd in P. nubifer larvae. The data presents mean values ± SD. Different letters denote significant differences (p < 0.05).

Figure 5.

The activity of catalase (CAT) (a) and Glutathione S-transferase (GST) (b) in P. nubifer larvae under Cd exposure. The data presents mean values ± SD. Different letters denote significant differences (p < 0.05).