Assessment of Acute and Multigenerational Toxicity of 1,3-Diphenylguanidine (DPG) on Freshwater Water Fleas: Oxidative Stress, Developmental, and Reproductive Effects

doi:10.21203/rs.3.rs-4304597/v1

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Assessment of Acute and Multigenerational Toxicity of 1,3-Diphenylguanidine (DPG) on Freshwater Water Fleas: Oxidative Stress, Developmental, and Reproductive Effects

https://doi.org/10.21203/rs.3.rs-4304597/v1

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1,3-Diphenylguanidine (DPG) is intensively used in the tire manufacturing industry as an accelerator, improving durability of rubber product. Despite its widespread use, concerns persist regarding the potential environmental risks associated with toxicological properties and mechanisms of DPG, remaining inadequately understood. This study aims to comprehensively assess the acute and multigenerational toxicity of DPG on freshwater water fleas (Moina macrocopa). We verified the acute toxicity of DPG by subjecting water fleas to varying concentrations and establishing the no-observed effect concentration (NOEC) for subsequent chronic exposure experiments.. Across four generations (P0, F1, F2, and F3), water fleas were continuously exposed to a concentration lower than the NOEC to investigate reproductive and developmental toxicity alongside oxidative stress indicators, including reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione disulfide (GSSG), and glutathione (GSH) activities. Multigeneration studies unveiled diminished reproduction rates, moulting, and body size across all exposed generations. Concurrently, heightened ROS levels coupled with decreased SOD, CAT, GSSG, and GSH activities signify the induction of antioxidant responses to DPG exposure. While reproductive effects were less pronounced in later generations, persistent oxidative stress across all generations underscores the necessity of multigenerational investigations in comprehending DPG's impact on water flea life cycles. This study underscores the necessity for additional research on DPG, promoting real-world exposure assessments and pragmatic experimental designs to precisely evaluate associated risks and implement sufficient environmental safeguards.

3-Diphenylguanidine

water flea

multigenerational

developmental

reproductive toxicity

oxidative stress

Substances originating from tire wear particles (TWPs), a byproduct of vehicle activity, are commonly carried away by stormwater runoff, spreading extensively and pervading diverse environments. (Pal et al., 2014;Spahr et al., 2020;Bakr et al.,2020). Recent research has shed light on the contamination of surface waters by anthropogenic chemicals derived from TWPs (Wagner et al. 2018; Jan Kole et al. 2017; Baensch-Baltruschat et al. 2020). Since the 1970s, researchers have been studying TWPs, but it was in the 1990s that research on road runoff started gaining momentum (Halle et al., 2020). These studies have revealed that chemicals used in tire manufacturing and their transformation products are transported into surface waters through untreated road runoff, significantly contributing to pollution in aquatic ecosystems (Unice et al., 2015; Seiwert et al., 2020). For example, hexamethoxymethylmelamine (HMMM), a tire cross-linking agent, and its transformation products have been identified in urban rivers (Hou et al., 2019). Furthermore, 1,3-Diphenylguanidine (DPG), a common compound found in TWP, has been detected in urban surface waters. Some studies have cautioned that the potential risks of DPG poses to the health and sustainability of aquatic ecosystems (Peter et al., 2018; Zahn et al., 2019).

DPG is a widely used compound in various industrial processes, particularly rubber vulcanization for tires, plastics, and leather products (Li and Zhao et al., 2023; Kannan, 2023b; ECHA, 2021; ECHA, 2020). The annual global production of DPG is estimated to reach around 11,100 tonnes. Asia seems to have the highest usage, with 5,300 tons per year, followed by the USA and Europe, with 2,400 tons per year each (Dos Santos, 2021; SDIS, 2002). More recent estimates suggest that Europe uses up to 10,000 metric tons of DPG annually (ECHA, 2018). Tire particles have been found to leach DPG into water samples worldwide, with concentrations ranging from very low ng/L to mg/L. (Seiwert et al., 2020; Zahn et al., 2019; Unice et al., 2015; Wik and Dave, 2009). Studies report DPG concentrations of 13 − 1,079 ng/L in Australian rivers, 5-540 ng/L in the USA, and 220 ng/L in Canada (Rauert et al., 2022; Hou et al., 2019; Johannessen et al., 2022b). European waters, encompassing the Rhine and its tributaries, demonstrated a median DPG concentration of 41 ng/L, with a peak concentration reaching 140 ng/L (Scheurer et al., 2021). Additional European investigations have identified DPG concentrations ranging from 5-100 ng/L (Zahn et al., 2019; Schulze et al., 2019), while Japanese waterways have recorded levels as high as 467 ng/L (Xie et al., 2021). These findings highlight the potential for aquatic organisms to encounter critical levels of DPG. Recognition of the acute toxicity of DPG is evident in abnormal behaviours observed in Oryzias latipes following exposure (Miti, 1992). A recent ecotoxicological study examined the hazardous effects of DPG found in tire wear leachate on fathead minnow embryos (Chibwe et al., 2022). While some investigations have revealed possible genotoxic effects in vitro (Dos Santos et al., 2022), the precise mechanism of action of DPG in aquatic organisms remains incompletely understood. To enhance comprehension of the long-term effects of DPG at environmentally relevant concentrations, conducting ecotoxicity studies using native species of freshwater organisms is imperative. Utilizing native species as bioindicators enhances ecological relevance and minimizes variations in toxicity associated with geographical disparities. This approach is crucial for understanding the potential impacts of DPG on aquatic ecosystems and effectively addressing logistical challenges while preventing the introduction of non-indigenous organisms into indigenous ecosystems.

Moina macrocopa, a vital zooplankton species, thrives in diverse tropical freshwater ecosystems, spanning regions such as Korea, Brazil, Malaysia, India, Mexico, Thailand, and the Philippines (Oh and Choi, 2012). Expanding the repertoire of test species is essential for comprehending species-specific responses to pollutants (Cui et al., 2018). Utilizing M. macrocopa in ecotoxicity studies presents numerous advantages, including ease of maintenance, sensitivity to pollutants, short life cycles, and global distribution. Moreover, its habitat in the upper layer of water bodies renders it particularly susceptible to aqueous pollutants (Engert et al., 2013). In this study, we utilized the advantages of water fleas to assess the toxicity of DPG. We focused on the effects of pollutants across multiple generations, as different generations may exhibit varying levels of toxicity and even adaptation to prolonged exposure (Kim et al., 2012; Lamichhane et al., 2014). Conducting multigenerational studies is crucial for accurately assessing ecological risks.

The aim was to initially confirm the acute toxicity of DPG in water fleas and subsequently investigate the chronic effects of exposure to environmentally relevant concentrations over four consecutive generations of test species. We evaluated various parameters such as moulting frequencies, total number of neonates, average number of neonates per brood, body length growth rate, first day of reproduction, and activities of ROS, CAT, SOD, GSH, and GSSG enzymes during the chronic exposure experiment.

Chemicals and test organism

DPG, sourced from Sigma-Aldrich (Saint Louis, United States), was used as this study standard material. A stock solution of DPG was prepared in Dimethylsulfoxide (DMSO) at a concentration of 10,000 mg/L and stored in an amber vial at -4℃ to prevent photodegradation. The test species, M. macrocopa, was obtained from the Korea Institute of Toxicology and maintained following USEPA recommendations (October, 2002a). Cultivation conditions included a constant ambient temperature of 20°C and a light/dark cycle of 16:8 hr, with the culture media prepared to a total hardness of 80–90 mg/L CaCO₃ (moderately hard water) as per EPA guidance. We added Selenium (Se) to the media and provided a daily diet of a blend of yeast, alfalfa, and Tetramin® (YCT) at a ratio of 1:1:1 with the green algae, Desmodesmus subspicatus to the water flea culture.

Acute toxicity test

We used neonates aged ≤ 24 hours for the acute toxicity tests. Each test group, including the carrying-solvent (DMSO) control, was replicated three times, with 10 individuals placed in beaker dishes containing 25 mL of the test solution. Test concentrations for DPG ranged from 1 to 1000 mg/L, with an additional control group. The assay was conducted over 48 hours at 22 ± 2°C, under a 16-hour light and 8-hour dark photoperiod. Renewal of the test medium was semi-statically done after 24 hours using freshly prepared stock solutions. We assessed the survival and immobility of the species after 24 and 48 hours of exposure. We defined immobility as the inability to swim after 15 seconds of light stimulation. We calculated LC₅₀ values using probit analysis and determined NOEC values using Dunnett's test.

Multigenerational test

The water flea reproduction test followed OECD 211 protocols and methods described by Barata et al. (2017). The exposure spanned four generations (P0, F1, F2, and F3) of water fleas, including a negative control group and a solvent control group. We exposed each generation to a nominal concentration of 0.5 mg/L DPG lower than the NOEC determined from acute toxicity testing. We isolated the first offspring of the F0 generation within 12 hours of birth and repeated this procedure for subsequent generations. During the experiment, we had 3 replicates for each control and treatment group, with one water flea per replicate individually exposed in beakers containing 30 mL of the test or control solutions. The photoperiod in the incubator was 16 hours of light and 8 hours of dark. The temperature was maintained at 20 ± 2°C. D. subspicatus (10⁶ cells/mL per replicate) was used to feed the water fleas, and the test solutions were replaced completely every 48 hours. The offspring were counted and discarded as described in the OECD guidelines (OECD 2012). Cultivation conditions and feeding regimen were consistent with acute toxicity testing. Parameters including neonates per water flea, average neonates per brood, the first day of reproduction, and molting frequency were recorded throughout the 14-day exposure period.

Biomarker experiments

The experiments for biomarker regulations were conducted in 1000 mL glass beakers filled with 300 mL of M4 medium and housing 60 water fleas each, allowing for three replicates per concentration along with a control group (n = 3). Following the protocol outlined for the multigenerational test, chronic exposure was administered to each water flea at a concentration of 0.5 mg/L for 14 days. Subsequently, 10 water fleas, randomly selected, underwent a thorough rinsing with deionized water before being transferred to 24-well plates. Then, we incubated them with 2,7-dichlorodihydrofluorescein diacetate (10 µM), a fluorescence agent, at 37°C for 30 minutes. To ensure accuracy and prevent potential in vitro interactions, we washed the individuals three times with 0.5 mL of a homogenization buffer using a portable vortex mixer. For the biochemical analyses, fluorescence intensity, measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively, via a microplate reader (Molecular Device, CA, USA), facilitated the quantification of ROS production in the test species.

Following exposure to 0.5 mg/L of DPG and the negative control, homogenates were obtained from pooled surviving water fleas in each beaker for more biomarker experiments. We prepared three independent replicates, each consisting of 50 individuals per treatment. To ensure accuracy and mitigate potential in vitro interactions, individuals underwent three washes with 0.5 mL of a homogenization buffer. This buffer, maintained at pH 7.4, comprised 100 mM potassium phosphate, 100 mM KCl, 1 mM EDTA, and 1:100 v/v of protease inhibitors in 1 mM dithiothreitol. Utilizing a motor pestle, individuals were homogenized, and resulting homogenates were centrifuged at 15,000 × g for 15 minutes at 4 ºC. The resulting supernatant was collected and immediately processed to determine SOD, CAT, GSH, and GSSG activities using spectrophotometric methods. All enzymatic activities were measured in triplicate for each homogenate pool. SOD activity quantified based on the production of superoxide anion, was measured at 470 nm, with findings reported in OD/unit. CAT activity was assessed by measuring the consumption of H₂O₂, with absorbance measured at 240 nm. Levels of GSH and GSSG were determined by measuring the reduced and oxidized states, respectively, at 412 nm for excitation and emission wavelengths, following the instructions provided by the manufacturer of the biomax assay and epigentex kits.

Statistical analysis

We determined LC₅₀ values through probit analysis using IBM's SPSS software. We calculated NOEC values using Dunnett's test. For statistical analysis, we employed GraphPad Prism 9.0 software. We used one-way ANOVA followed by Tukey's post hoc test to compare means among different groups. Additionally, we conducted Pearson correlation analysis using GraphPad Prism 9.0 software to explore potential correlations between parameters in the water flea.

Acute toxicity effects

The survival probability curve for M. macrocopa demonstrates a trend dependending on both concentration and duration of exposure. All test species were found to be 100% viable in the control group. However, with increasing concentrations of DPG and longer exposure times (24 and 48 hours), there was a significant decrease in survival rates (p < 0.05), as illustrated in Fig. 1. We observed mortality across concentrations ranging from 5 to 1000 mg/L (Fig. 1). The estimated 24-h and 48-h LC₅₀, as well as the NOEC of DPG, were found to be 35.0 mg/L, 13.7 mg/L, and 1.0 mg/L, respectively.

Multigenerational Chronic effects on growth and reproduction

The chronic effects of DPG on the growth and reproduction of M. macrocopa were investigated across multiple generations. No deaths or ephippia were observed throughout the experiment in any treatment groups. The number of neonates produced per water flea did not exhibit significant differences among the treatments, irrespective of the generation (P0, F1, F2, F3) (Fig. 2, Table S1-S4). However, in the P0 generation, the number of offspring decreased after the exposure compared to the control group (Fig. 2a). The data reveals a consistent trend of higher neonate counts in the control group compared to the treated group across all four generations. The average number of neonates per brood was decreased in the exposed groups over all generations but was not statically evident. As shown in the number of neonates per water flea, the number per brood slightly increased across generations (Fig. 3a). Nevertheless, this increase was not statistically significant (P > 0.05). The appearance of the first offspring occurred after 5 days in all generations exposed to DPG, as well as in the control group (Fig. 3b). Continuous exposure significantly impacted the body length of water fleas across generations compared to the control group (P < 0.05) (Fig. 3c). Moreover, the number of moulting for the exposed groups exhibited the significant decrease over all generations (P < 0.05). Still, the level showed a tendency to recover (Fig. 3d).

ROS activity induction

We measured ROS activity across generations to evaluate the extent of oxidative cellular damage induced by chronic exposure to DPG (Fig. 4). In the P0 generation, the exposed individuals exhibited a significant (p < 0.05) increase in free radical formation by 21.5% compared to the control. Similarly, in the F1, F2, and F3 generations, ROS levels showed a notable increase following DPG exposure, by 20.92%, 20.71%, and 20.3%, respectively, compared to the control (p > 0.05).

Antioxidant regulations

DPG exposure resulted in a reduction in CAT activity of water fleas. Despite the apparent difference in the average values between the control and the exposure groups, the decrease was not statistically significant in the P0, F1, and F3 generations (P > 0.05) (Fig. 5a). Yet, in the F2 generation, the enzyme levels significantly decreased in the DPG treated group (P < 0.05). Moreover, in the F2 generation, there was a significant difference in CAT activity (P < 0.05) compared to control. The DPG-treated groups significantly reduced SOD levels in all generations. Among the exposed group, the levels were significantly higher in the F3 generation, possibly indicating the recovery in the enzyme activity (Fig. 5b). GSH activity varied among the generations. We observed apparent reductions in GSH activity for the exposed groups in all generations. However, the decrease in the F2 generation was not statistically significant (P > 0.05). The P0 generation exhibited the maximum decrease in GSH activity (P < 0.05) compared to other generations (Fig. 5c). In the P0, F1, and F3 generations, GSSG levels significantly decreased in the DPG-treated groups. Meanwhile, in the F3 generation, GSSG levels were significantly higher in the treated group than those affected by the exposures in other generations (Fig. 5d).

Relationships between physiological and biochemical parameters

Pearson correlation analysis was performed to explore the relationships among physiological and biochemical parameters in the four generations (P0, F1, F2, and F3). The analysis unveiled significant correlations between various endpoints, indicating that exposure to DPG resulted in multiple adverse effects on the test species. Specifically, the results demonstrated a significant positive correlation between ROS production and concentration (0.91, P < 0.05). Conversely, the average number of neonates (-0.97), moulting (-0.93), size (-0.99), CAT (-0.64), GSH (-0.98), GSSG (-0.99), and SOD (-0.97) exhibited negative correlations with the concentration of DPG in the water fleas (Fig. 6, Table S5). When considering the limited availability of data points at a single concentration, it is important to acknowledge the inherent limitations in establishing a robust correlation. The analysis may provide insights into the relationship between the concentration and various factors. However, it is important to note that we should interpret the findings cautiously due to the lack of a comprehensive dataset. These preliminary observations can serve as a basis for further investigation and highlight the need for additional data to strengthen the validity of the correlation analysis.

Acute and multigenerational effects

The release of DPG into aquatic ecosystems has been escalating, primarily attributable to its increased usage (Zhang et al., 2023). Consequently, there has been a pressing need to assess the impacts of DPG on aquatic organisms. This study aimed to investigate the short-term and long-term effects of DPG on water fleas, focusing on various biochemical and physiological endpoints. The assessment of acute toxicity with LC₅₀ values revealed the sensitivity of water fleas to DPG. We investigated the chronic and multigenerational impacts on water flea species by measuring growth and reproduction rate. In vivo experiments conducted over four generations of water fleas revealed that exposure to environmentally relevant concentrations of DPG induced changes in their body length and reproductive parameters. The results indicated that DPG exposure inhibited population growth-related endpoints, as depicted in Figs. 2 and 3. Previous studies have demonstrated that DPG can cause skin and eye irritation in humans (Al-Maliki et al., 2021) and induce reproductive toxicity in animal studies (Koëter et al., 1992).

Interestingly, continuous exposure of water fleas to 0.5 mg/L of DPG appeared to prompt adaptation, the negative effects on reproduction in subsequent generations. Jeong et al. (2016) observed a comparable outcome when studying the effects of PFOS on Daphnia magna, wherein subsequent generations exhibited a diminished negative impact on reproduction. Studying the long-term effects of DPG toxicity across multiple generations poses a significant challenge. Due to limited research on multigenerational impacts, it is difficult to identify a clear pattern of harmful effects on fecundity and understand the adaptation phenomenon. To better understand the intergenerational changes and reproductive impairment caused by DPG, further comprehensive studies are needed.

The early developmental stages of water fleas play a crucial role in hormonal regulation and normal growth. For example, hormones such as ecdysteroids and sesquiterpenoids control molting processes, essential for developmental transitions and growth in cladocerans (Adhitama et al., 2020). While information regarding the effects of DPG on hormonal regulation in aquatic crustaceans remains limited, our findings suggest that exposure to DPG during the early developmental stages of water fleas has the potential to disrupt the intermoult period, resulting in significant growth retardation across four generations. Despite the scarcity of multigenerational studies exploring the toxicity of DPG and the limited data on intergenerational changes in fecundity provided by existing studies, further investigation is necessary to elucidate the observed adaptation phenomenon in reproductive impairment. Nevertheless, our results demonstrate significant adverse effects on physiological and lifespan parameters in water fleas exposed to DPG, particularly in the initial generation (P0). We hypothesized that the reduction in neonates and growth inhibition may be associated with accumulating biochemical and physiological stress over multiple generations. Therefore, additional biochemical markers have been measured in this study to assess the negative effects of DPG exposure on the cellular defence and tolerance mechanisms of water fleas.

Increased oxidative stress

Water fleas exposed to DPG exhibited intracellular oxidative stress, which resulted in considerable modifications of the antioxidant defence system and cellular stress response enzymes. DPG exposure lowered antioxidant enzyme activity across all generations. These results were in accordance with other research (Varó et al. 2019; Eom et al. 2020; Lee et al. 2021; Suman et al., 2020), which demonstrated the considerable influence on the oxidative status of cladocerans and other zooplankton species. The increased oxidative stress and alterations in the antioxidant defence system are likely accountable for the observed reductions in growth and reproduction parameters across generations. Notably, the P0 generation exhibited a more pronounced decrease in the antioxidant defence system compared to the other generations, potentially indicating damage to the antioxidant system. These results underscore the detrimental effects of DPG on water fleas and emphasize the importance of comprehending the biochemical mechanisms underlying oxidative stress in response to environmental pollutants.

Reactive oxygen species (ROS) are highly reactive molecules that induce oxidative damage in cells, encompassing superoxide anion, hydrogen peroxide, hydroxyl radical, and singlet oxygen (Bhattacharya 2015). Oxidative stress arises from an imbalance between ROS production and the cell's ability to detoxify or repair the resulting damage (Mena et al., 2009). Certain environmental pollutants are recognized contributors to oxidative stress (Naidoo and Birch-Machin 2017), with excessive ROS levels leading to the oxidation of lipids, proteins, DNA, and other cellular components (Kim et al., 2012). While ROS plays crucial roles in cell signaling, immune response, and cellular regulation at low levels, an excessive increase can result in oxidative damage (Li et al., 2017). Increased ROS levels can have varied effects on antioxidant enzymes such as SOD, CAT, GSSG, and GSH. The presence of ROS can create survival pressure, potentially selecting offspring with stronger anti-stress abilities over multiple generations (Schultz et al., 2016). Interestingly, water fleas gradually reduced oxidative stress across successive generations, suggesting enhanced tolerance to DPG-induced damage through antioxidative responses (Kaletsky and Murphy, 2010; Olsen and Gill, 2017). SOD, a critical antioxidant enzyme neutralizing superoxide radical, may exhibit decreased activity when ROS levels surge beyond its capacity to neutralize effectively (Kwiecien et al., 2014; Ercal et al., 2001). Our findings indicate reduced SOD activity in all generations of water fleas following exposure to DPG (P0, F1, F2, and F3). This reduction likely stems from the breakdown of antioxidant defence mechanisms after prolonged DPG exposures, as observed after 14 days of exposure. Similarly, CAT, responsible for converting harmful hydrogen peroxide into water and oxygen, can see compromised activity under increased ROS levels (Weydert and Cullen 2010; Lin et al., 2021). Our study reveals reduced CAT activity in all four generations of water fleas (P0, F1, F2, and F3) following exposure to DPG.

Glutathione, a potent antioxidant, is pivotal in shielding cells against oxidative damage, primarily by scavenging ROS (Jena et al., 2023). However, when ROS levels escalate, the availability of reduced GSH may diminish due to its utilization in neutralizing ROS, consequently leading to reduced GSH levels (Cheng et al., 2021). This depletion can compromise the antioxidant defence system, increasing the likelihood of oxidative stress occurrence. Aquatic organisms exposed to oxidative stressors may witness a decrease in glutathione disulfide (GSSG) levels and an increase in ROS (Xia et al., 2016). Elevated ROS levels can disrupt the cellular antioxidant defence system, potentially overwhelming the organism's capacity to convert GSSG to its reduced form, GSH (Massarsky et al., 2017). This shift towards increased ROS and decreased GSSG indicates oxidative stress in aquatic organisms, which can have detrimental effects on cellular function and overall health. Therefore, we examined GSSG and GSH levels in control and DPG-treated water fleas after 14 days of exposure. The analysis revealed a decrease in GSSG and GSH levels after exposure to DPG in all four generations (P0, F1, F2, and F3). Although alterations in antioxidant enzyme activities can provide indirect evidence of oxidative stress, they may be insufficient in determining the extent of oxidative challenge posed by DPG. In this study, we observed a disruption in the redox-balance system of water fleas in response to DPG, indicating their heightened sensitivity to this compound (Fig. 7).

The results indicate a slight but statistically significant increase in the levels of SOD, CAT, GSH, and GSSG content in the F3 generation, suggesting a potential enhancement of the antioxidant defence system compared to the preceding generations. F3 generation developed a stronger antioxidant defence system, which could help reduce potential harm. According to Drevet et al. (2006) and Kalmar and Greensmith (2009), these enhancements in antioxidant and cytoprotective phase II detoxification enzyme activities suggest a progressive reinforcement of water fleas' biochemical capacity to scavenge ROS and detoxify substances. Previous research has often observed a simultaneous increase in antioxidant enzyme activities and glutathione (GSH) consumption in response to environmental pollutants during short-term exposure (Muthuraman et al., 2014; Rico et al., 2015; Zhu et al., 2011). However, most studies on the underlying mechanisms have been limited to single-generation experiments. The findings of our study indicate increases in the levels of SOD, CAT, GSSG, and GSH enzymes in subsequent generations of water fleas. These results demonstrate, for the first time, that the biochemical anti-stress mechanism of water fleas gradually strengthens after multigenerational exposure to DPG, potentially accounting for changes in multigenerational features such as increased neonate numbers and moulting. Further research and investigation are necessary to fully understand the impact of oxidative stress on organisms and elucidate the molecular mechanisms underlying oxidative stress.

In acute toxicities studies as the concentration and duration of DPG exposure increased, the survival rate of water fleas decreased. The LC₅₀ values for 24 hours and 48 hours were 35.0 mg/L and 13.7 mg/L, and NOEC of DPG, were found to be 1.0 mg/L respectively. In the multigenerational study, we did not observe any deaths, and there were no significant differences in the number of neonates per water flea among the treatment groups. However, we observed a decrease in the number of offspring in the P0 generation exposed to a concentration lower than the NOEC. The increase in the number of neonates as the generation number rises is intriguing. The chronic exposure of water fleas to DPG over multiple generations resulted in increased ROS activity across all generations, with significant increases observed in the P0, F1, F2, and F3 generations. Additionally, the antioxidant response, as measured by CAT and SOD activity, exhibited variations among the generations. The F3 generation of water fleas showed enhancement compared to previous generations, indicating a progressive reinforcement of their ability to scavenge ROS and detoxify over time. It is noteworthy that the levels of SOD, CAT, GSSG, and GSH enzymes showed sustained elevation in subsequent generations, potentially explaining positive changes in features such as the increased number of neonates and moulting. This study provides insights into the biochemical responses of water fleas to 14-day DPG exposure. It contributes biochemical evidence supporting the notion of adaptive effects resulting from multigenerational DPG exposure in water fleas.

Credit authorship contribution statement

Thodhal Yoganandham Suman: Conceptualization, Methodology, Formal analysis, Data Curation, Writing – Original Draft. Soo-Yeon Kim: Conceptualization, Methodology, Validation, Software, Project administration. Younghoon Jang: Methodology, Validation, Software. Junho Jeon: Conceptualization, Methodology, Validation, Manuscript revision, Project administration, Supervision, Funding acquisition.

Credit authorship contribution statement

Thodhal Yoganandham Suman: Conceptualization, Methodology, Formal analysis, Data Curation, Writing – Original Draft. Soo-Yeon Kim: Conceptualization, Methodology, Validation, Software, Project administration. Younghoon Jang: Methodology, Validation, Software. Junho Jeon: Conceptualization, Methodology, Validation, Manuscript revision, Project administration, Supervision, Funding acquisition.

Funding

This work was supported by Korea Environment Industry & Technology Institute (KEITI) through Advanced Technology Development Project for Predicting and Preventing Chemical Accidents Project, funded by Korea Ministry of Environment (MOE) (GM16040) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education(2021R1A2C2008529).

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

The authors have no relevant financial or non-financial interests to disclose.

All authors have read, understood, and have complied as applicable with the statement on "Ethical responsibilities of Authors" as found in the Instructions for Authors and are aware that with minor exceptions, no changes can be made to authorship once the paper is submitted"."

Ethics approval Not applicable

Consent to participate Not applicable

Consent for publication

The manuscript is approved by all authors for publication.

Competing interests

The authors declare no competing interests

Acknowledgments

We thank Gyeongnam Branch Institute, Korea Institute of Toxicology (KIT), Jinju-si 52834, Republic of Korea for providing necessary research facility throughout the study. This work was supported by Korea Environment Industry & Technology Institute (KEITI) through Advanced Technology Development Project for Predicting and Preventing Chemical Accidents Project, funded by Korea Ministry of Environment (MOE) (GM16040) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education(2021R1A2C2008529).

Adhitama N, Kato Y, Matsuura T, Watanabe H (2020) Roles of and cross-talk between ecdysteroid and sesquiterpenoid pathways in embryogenesis of branchiopod crustacean Daphnia magna. PLoS ONE 15:0239893
Al-Maliki HSJ, Al-Jaberi RAF, Imeer ATA, Lafta GA, Kadhum AAH, Allami AJ, Al-Rekabi RAHEMRM, Al-Gazally SHA, Alasadi ME, Al-Qanbar YF MH (2021) Skin Allergies Generated by Rubber Gloves and Excessive Soaps and Sterilizers due to COVID-19 Pandemic: a Review. Lat Am J Pharm 317–324
Baensch-Baltruschat B, Kocher B, Stock F, Reifferscheid G (2020) Tyre and road wear particles (TRWP)-A review of generation, properties, emissions, human health risk, ecotoxicity, and fate in the environment. Sci Total Environ 733:137823
Bakr AR, Fu GY, Hedeen D (2020) Water quality impacts of bridge stormwater runoff from scupper drains on receiving waters: A review. Sci Total Environ 726:138068
Barata C, Campos B, Rivetti C, LeBlanc GA, Eytcheson S, McKnight S, Tobor-Kaplon M, de Vries Buitenweg S, Choi S, Choi J, Sarapultseva EI (2017) Validation of a two-generational reproduction test in Daphnia magna: an interlaboratory exercise. Sci Total Environ 579:1073–1083
Bhattacharya S (2015) Reactive oxygen species and cellular defense system. Free radicals Hum health disease, 17–29
Cheng X, Xu HD, Ran HH, Liang G, Wu FG (2021) Glutathione-depleting nanomedicines for synergistic cancer therapy. ACS Nano 15:8039–8068
Chibwe L, Parrott JL, Shires K, Khan H, Clarence S, Lavalle C, Sullivan C, O'Brien AM, De Silva AO, Muir DC, Rochman CM (2022) A deep dive into the complex chemical mixture and toxicity of tire wear particle leachate in fathead minnow. Environ Toxicol Chem 41:1144–1153
Cui R, Kwak J, An YJ (2018) Comparative study of the sensitivity of Daphnia galeata and Daphnia magna to heavy metals. Ecotoxicol Environ Saf 162:63–70
Dos Santos MM, Cheriaux C, Jia S, Thomas M, Gallard H, Croué JP, Carato P, Snyder SA (2022) Genotoxic effects of chlorinated disinfection byproducts of 1, 3-diphenylguanidine (DPG): Cell-based in-vitro testing and formation potential during water disinfection. J Hazard Mater 436:129114
Drevet JR (2006) The antioxidant glutathione peroxidase family and spermatozoa: a complex story. Mol Cell Endocrinol 250:70–79
ECHA 1,3-di-o-tolylguanidine (EC Number (2018) 202-577-6 | CAS Number: 97-39-2) REACH Dossier. European Chemicals Agency
ECHA, (2020) 1,3-diphenylguanidine (EC Number: 203-002-1 | CAS Number: 102-06-7) REACH Dossier. European Chemicals Agency. https://echa.europa.eu/documents/10162/4df27360-03aa-3c93-54f0-08f8366f42f3 (Accessed August 2023)
ECHA (2021) 1,3-di-o-tolylguanidine (EC Number: 202-57-6 | CAS Number: 97-39-2) REACH Dossier. European Chemicals Agency. https://echa.europa.eu/substance- information/-/substanceinfo/100.002.344 (Accessed August 2023)
Engert A, Chakrabarti S, Saul N, Bittner M, Menzel R, Steinberg CEW (2013) Interaction of temperature and an environmental stressor: Moina macrocopa responds with increased body size, increased lifespan, and increased offspring numbers slightly above its temperature optimum. Chemosphere 90:2136–2141
Eom HJ, Nam SE, Rhee JS (2020) Polystyrene microplastics induce mortality through acute cell stress and inhibition of cholinergic activity in a brine shrimp. Mol Cell Toxicol 16:233–243
Ercal N, Gurer-Orhan H, Aykin-Burns N (2001) Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage. Curr Top Med Chem 1:529–539
Halle LL, Palmqvist A, Kampmann K, Khan FR (2020) Ecotoxicology of micronized tire rubber: Past, present and future considerations. Sci Total Environ 706:135694
Hou F, Tian Z, Peter KT, Wu C, Gipe AD, Zhao H, Alegria EA, Liu F, Kolodziej EP (2019) Quantification of organic contaminants in urban stormwater by isotope dilution and liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 411:7791–7806
Jena AB, Samal RR, Bhol NK, Duttaroy AK (2023) Cellular Red-Ox system in health and disease: The latest update. Biomed Pharmacother 162:114606
Jeong TY, Yuk MS, Jeon J, Kim SD (2016) Multigenerational effect of perfluorooctane sulfonate (PFOS) on the individual fitness and population growth of Daphnia magna. Sci Total Environ 569:1553–1560
Johannessen C, Parni JM (2021b) Environmental modelling of hexamethoxymethylmelamine, its transformation products, and precursor compounds: An emerging family of contaminants from tire wear. Chemosphere 280:130914
Kaletsky R, Murphy CT (2010) The role of insulin/IGF-like signaling in C. elegans longevity and aging. Dis Models Mech 3:415–419
Kalmar B, Greensmith L (2009) Induction of heat shock proteins for protection against oxidative stress. Adv Drug Deliv Rev 61:310–318
Kim HY, Lee MJ, Yu SH, Kim SD (2012) The individual and population effects of tetracycline on Daphnia magna in multigenerational exposure. Ecotoxicology 21:993–1002
Kim YJ, Kim EH, Hahm KB (2012) Oxidative stress in inflammation-based gastrointestinal tract diseases: Challenges and opportunities. Can J Gastroenterol Hepatol Can 27:1004–1010
Koëter HBWM, Regnier JF, Van Marwijk MW (1992) Effect of oral administration of 1, 3-diphenylguanidine on sperm morphology and male fertility in mice. Toxicology 71:173–179
Kole PJ, Löhr AJ, Van Belleghem FG, Ragas AM (2017) Wear and tear of tyres: a stealthy source of microplastics in the environment. Int J Environ Res Public Health 14:1265
Kwiecien S, Jasnos K, Magierowski M, Sliwowski Z, Pajdo R, Brzozowski B, Mach T, Wojcik D, Brzozowski T (2014) Lipid peroxidation, reactive oxygen species and antioxidative factors in the pathogenesis of gastric mucosal lesions and mechanism of protection against oxidative stress-induced gastric injury. J Physiol Pharmacol 65:613–622
Lamichhane K, Garcia SN, Huggett DB, DeAngelis DL, La Point TW (2014) Exposures to a selective serotonin reuptake inhibitor (SSRI), sertraline hydrochloride, over multiple generations: changes in life history traits in Ceriodaphnia dubia. Ecotoxicol Environ Saf 101:124–130
Lee DH, Lee S, Rhee JS (2021) Consistent exposure to microplastics induces age-specific physiological and biochemical changes in a marine mysid. Mar Pollut Bull 162:111850
Li Z, Xu X, Leng X, He M, Wang J, Cheng S, Wu H (2017) Roles of reactive oxygen species in cell signaling pathways and immune responses to viral infections. Arch Virol 162:603–610
Li ZM, Kannan K (2023) Occurrence of 1,3-diphenylguanidine, 1,3-di-o-tolylguanidine, and 1,2,3-triphenylguanidine in indoor dust from 11 countries: implications for human exposure. Environ Sci Technol 57:6129–6138
Lin Y, Lin Y, Lin M, Fan Z, Lin H (2021) Influence of hydrogen peroxide on the ROS metabolism and its relationship to pulp breakdown of fresh longan during storage. Food Chem X 12:100159
Massarsky A, Kozal JS, Di Giulio RT (2017) Glutathione and zebrafish: Old assays to address a current issue. Chemosphere 168:707–715
Mena S, Ortega A, Estrela JM (2009) Oxidative stress in environmental-induced carcinogenesis. Mutat Research/Genetic Toxicol Environ Mutagen 674:36–44
Methods for Measuring the Acute Toxicity of Effluents and (2002a) Receiving Waters to Freshwater and Marine Organisms, vol 20460. USEPA (October, Washington, DC
MITI (1992) : Supervision of Chemical Products Safety Division, Basic Industries Bureau MITI, Ed. by CITI 1992: Biodegradation and Bioaccumulation, Data of Existing Chemicals Based on CSCL Japan (publication), Published by Japan Chemical Industry Ecology-Toxicology & Information Center. Biodegradation and Bioaccumulation Data of Existing Chemicals Based on the CSCL Japan, Japan Chemical Industry Ecology-Toxicology & Information Center
Muthuraman P, Ramkumar K, Kim DH (2014) Analysis of dose-dependent effect of zinc oxide nanoparticles on the oxidative stress and antioxidant enzyme activity in adipocytes. Appl Biochem Biotechnol 174:2851–2863
Naidoo K, Birch-Machin MA (2017) Oxidative stress and ageing: the influence of environmental pollution, sunlight and diet on skin. Cosmetics 4:4
OECD (2012) Guideline for the Testing of Chemicals No 211. Daphnia magna Reproduction Test. Organization for Economic Cooperation and development, Paris, p 202
Oh S, Choi K (2012) Optimal conditions for three brood chronic toxicity test method using a freshwater macroinvertebrate Moina macrocopa. Environ Monit Assess 184:3687–3695
Olsen A, Gill MS (2017) Ageing: Lessons from C. elegans. Springer, Cham
Pal A, He Y, Jekel M, Reinhard M, Gin KYH (2014) Emerging contaminants of public health significance as water quality indicator compounds in the urban water cycle. Environ Int 71:46–62
Peter KT, Tian Z, Wu C, Lin P, White S, Du B, McIntyre JK, Scholz NL, Kolodziej EP (2018) Using high-resolution mass spectrometry to identify organic contaminants linked to urban stormwater mortality syndrome in coho salmon. Environ Sci Technol 52:10317–10327
Rauert C, Charlton N, Okoffo ED, Stanton RS, Agua AR, Pirrung MC, Thomas KV (2022) _ Concentrations of tire additive chemicals and tire road wear particles in an Australian urban tributary. Environ Sci Technol 56:2421–2431
Rico CM, Peralta-Videa JR, Gardea-Torresdey JL (2015) Chemistry, Biochemistry of Nanoparticles, and Their Role in Antioxidant Defense System in Plants. Nanotechnology and Plant Sciences. Springer, Cham, p 1e17
Scheurer M, Sandholzer A, Schnabel T, Schneider-Werres S, Schaffer M, B¨ornick H, Beier S (2021) Persistent and mobile organic chemicals in water resources: occurrence and removal options for water utilities. Water Supply 22:1575–1592
Schultz CL, Wamucho A, Tsyusko OV, Unrine JM, Crossley A, Svendsen C, Spurgeon DJ (2016) Multigenerational exposure to silver ions and silver nanoparticles reveals heightened sensitivity and epigenetic memory in Caenorhabditis elegans. Proc R Soc Ser B Biol Sci 283:20152911
Schulze S, Zahn D, Montes R, Rodil R, Quintana JB, Knepper TP, Reemtsma T, Berger U (2019) Occurrence of emerging persistent and mobile organic contaminants in European water samples. Water Res 153:80–90
SDIS Initial Assessment report For SIAM 14: 1,3-diphenylguanidine. France 2002, Paris
Seiwert B, Klöckner P, Wagner S, Reemtsma T (2020) Source-related smart suspect screening in the aqueous environment: search for tire-derived persistent and mobile trace organic contaminants in surface waters. Anal Bioanal Chem 412:4909–4919
Spahr S, Teixidó M, Sedlak DL, Luthy RG (2020) Hydrophilic trace organic contaminants in urban stormwater: occurrence, toxicological relevance, and the need to enhance green stormwater infrastructure. Environ Sci Water Res Technol 6:15–44
Suman TY, Jia PP, Li WG, Junaid M, Xin GY, Wang Y, Pei DS (2020) Acute and chronic effects of polystyrene microplastics on brine shrimp: First evidence highlighting the molecular mechanism through transcriptome analysis. J Hazard Mater 400:123220
Unice KM, Bare JL, Kreider ML, Panko JM (2015) Experimental methodology for assessing the environmental fate of organic chemicals in polymer matrices using column leaching studies and OECD 308 water/sediment systems: application to tire and road wear particles. Sci Total Environ 533:476–448
Unice KM, Bare JL, Kreider ML, Panko JM (2015) Experimental methodology for assessing the environmental fate of organic chemicals in polymer matrices using column leaching studies and OECD 308 water/sediment systems: application to tire and road wear particles. Sci Total Environ 533:476–487
Varó I, Perini A, Torreblanca A, Garcia Y, Bergami E, Vannuccini ML, Corsi I (2019) Time-dependent effects of polystyrene nanoparticles in brine shrimp Artemia franciscana at physiological, biochemical and molecular levels. Sci Total Environ 675:570–580
Wagner S, Hüffer T, Klöckner P, Wehrhahn M, Hofmann T, Reemtsma T (2018) Tire wear particles in the aquatic environment-a review on generation, analysis, occurrence, fate and effects. Water Res 139:83–100
Weydert CJ, Cullen JJ (2010) Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue. Nat Protoc 5:51–66
Wik A, Dave G (2009) Occurrence and effects of tire wear particles in the environment – a critical review and an initial risk assessment. Environ Pollut 157:1–11
Xia L, Chen S, Dahms HU, Ying X, Peng X (2016) Cadmium induced oxidative damage and apoptosis in the hepatopancreas of Meretrix meretrix. Ecotoxicol:959–969
Xie L, Nakajima F, Kasuga I, Kurisu F (202) Simultaneous screening for chemically diverse micropollutants in public water bodies in Japan by high-performance liquid chromatography–Orbitrap mass spectrometry. Chemosphere 273: 128524
Zahn D, Mucha P, Zilles V, Touffet A, Gallard H, Knepper TP, Frömel T (2019) Identification of potentially mobile and persistent transformation products of REACH-registered chemicals and their occurrence in surface waters. Water Res 150:86–96
Zahn D, Mucha P, Zilles V, Touffet A, Gallard H, Knepper TP, Fr¨omel T (2019) Identification of potentially mobile and persistent transformation products of REACH-registered chemicals and their occurrence in surface waters. Water Res 150:86–96
Zhang HY, Huang Z, Liu YH, Hu LX, He LY, Liu YS, Zhao JL, Ying GG (2023) Occurrence and risks of 23 tire additives and their transformation products in an urban water system. Environ Int 171:107715
Zhao HN, Hu X, Gonzalez M, Rideout CA, Hobby GC, Fisher MF (2023) Screening pphenylenediamine antioxidants, their transformation products, and industrial chemical additives in crumb rubber and elastomeric consumer products. Environ Sci Technol 57:2779–2791
Zhu X, Zhou J, Cai Z (2011) The toxicity and oxidative stress of TiO2 nanoparticles in marine abalone (Haliotis diversicolor supertexta). Mar Pollut Bull 63:334–338

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Assessment of Acute and Multigenerational Toxicity of 1,3-Diphenylguanidine (DPG) on Freshwater Water Fleas: Oxidative Stress, Developmental, and Reproductive Effects

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Abstract

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Introduction.

Material and methods

Chemicals and test organism

Acute toxicity test

Multigenerational test

Biomarker experiments

Statistical analysis

Results

Acute toxicity effects

Multigenerational Chronic effects on growth and reproduction

ROS activity induction

Antioxidant regulations

Relationships between physiological and biochemical parameters

Discussion

Acute and multigenerational effects

Increased oxidative stress

Conclusions

Credit authorship contribution statement

Declarations

References

Supplementary Files

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