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Article

Assessment of Growth and Physiological Responses of Lemna minor Exposed to 4-Aminodiphenylamine, a Tire Wear Compound

by
Shila Kandel
,
Naja’Ree Campbell
,
Abubakar Abdulkadir
,
Kristin Moore
,
Raphyel Rosby
and
Ekhtear Hossain
*
Department of Biological Sciences and Chemistry, Southern University and A&M College, Baton Rouge, LA 70813, USA
*
Author to whom correspondence should be addressed.
Pollutants 2025, 5(3), 20; https://doi.org/10.3390/pollutants5030020
Submission received: 10 May 2025 / Revised: 23 June 2025 / Accepted: 1 July 2025 / Published: 7 July 2025
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Abstract

4-Aminodiphenylamine (4-ADPA) is a common additive in rubber tires, known for its antioxidant properties. It plays a crucial role in enhancing tire durability by preventing issues such as drying, cracking, and degradation from prolonged exposure to environmental factors like heat, oxygen, and ozone. However, despite its advantages in extending tire lifespan, the use of 4-ADPA raises significant environmental concerns. As tires wear down, microscopic tire wear particles (TWPs) containing 4-ADPA are released into the environment with substantial leaching, contaminating the waterways. The 4-ADPA leachates pollute and pose a threat to aquatic ecosystems, affecting various forms of marine life. The current study investigates the ecotoxicological effects of 4-ADPA on the aquatic plant Lemna minor (L. minor), focusing on its impact on relative growth and physiological biomarkers. Several parameters were assessed to evaluate ecotoxicity, including frond morphology, fresh biomass, total frond number, chlorophyll content, and starch accumulation. L. minor was grown for 7 and 14 days under controlled laboratory conditions using Hoagland media with varying concentrations of 4-ADPA (10–100 μg/L), while a control group was maintained in media without 4-ADPA. The results indicate that exposure to 4-ADPA led to a dose-dependent reduction in fresh biomass, total frond number, and chlorophyll levels. Lugol’s staining revealed increased starch accumulation in the fronds after exposure to 4-ADPA. The biological effects observed in L. minor following exposure to 4-ADPA, even at environmentally relevant concentrations, demonstrate a significant ecotoxicological impact on aquatic ecosystems. Further research involving additional species and investigating the mechanisms behind 4-ADPA toxicity is recommended to better understand its long-term consequences.

Graphical Abstract

1. Introduction

With increased urbanization, the reliance on cars has increased, and persistent micro-pollution from car tires is inevitable [1,2]. Car tires possess a complex chemical composition, including polymeric material, fillers, softeners, vulcanization agents, and other additives that increase their strength, mechanical properties, and durability [3,4]. 4-Aminodiphenylamine (4-ADPA) is a common additive in rubber tires, known for its antioxidant properties. It plays a vital role in enhancing tire durability by preventing issues such as drying, cracking, and degradation from prolonged exposure to environmental elements like heat, oxygen, and ozone [3,5]. As car tires wear down because of high friction on the roadway, microscopic particles, often called tire wear particles (TWPs), are released into the atmosphere, leading to environmental pollution [1]. A few recent studies on rubber tires and tire wear particles have identified 4-ADPA as a TWP that can have ecological and toxicological consequences. The 4-ADPA, a common antioxidant used in tire manufacturing, was found in all airborne particulate matter samples collected along a highway in Mississippi, USA [2]. In a study of two cities in Germany, Kuntz et al. confirmed the presence of 4-ADPA in urban particulate matter aerosol [6]. Chemical profiling and suspect screening by McMinn et al. identified the presence of 4-ADPA in crumb rubber extracts [7]. These studies confirm that many TWPs with varying concentrations are present in urban aerosols and potentially carcinogenic and genotoxic to humans [6]. The environmental residue of 4-ADPA from tire wear is washed away by rain and released into the aquatic environment through urban wastewater runoff. Many of these particles do not degrade and persist in the aquatic ecosystem, and even bioaccumulate in aquatic life forms, posing threats to various forms of marine life. Various environmental studies have demonstrated the toxicity of TWPs on marine life forms [3]. The toxicological studies in aquatic organisms exhibited bioaccumulation of these compounds, and long-term exposure causes growth and developmental damage, neurotoxicity, respiratory, reproductive, and intestinal toxicity, and multi-organ failure [8,9,10]. Owing to the ecotoxicological effects on aquatic organisms, TWP-derived compounds have gained increasing attention in environmental health science research.
The free-floating plants growing on the water surface are called duckweeds, comprising monocotyledonous macrophytes [11,12]. Among the family Lemnaceae of this class, the Lemna genus includes 14 species of floating plants. The species Lemna minor (L. minor) is probably the best known of the genus, also known as common duckweed, and is usually found in still or slow-moving waters [13]. It is widely available in the aquatic ecosystems of Asia, Africa, Europe, and North America, including the United States and Canada [11]. It has a simple morphology, faster growth, and a faster reproduction rate than other vascular aquatic species [14]. Aquatic macrophytes are of ecological significance, as they are essential in nutrient cycling, sediment stabilization, oxygenation, and in providing food and marine habitat. They have various applications in agriculture, pharmaceuticals, phytoremediation, and energy production, and they represent higher aquatic plants in research. The Lemna species offer several advantages as a study model in physiological and ecotoxicological research, since they are adapted to different climatic conditions, grow rapidly, reproduce asexually with homogeneity, are easy to maintain in laboratory cultures, and are sensitive to toxicants [12,15]. The developmental biomarkers, such as growth parameters and physiological biomarkers like chlorosis and pigment content, are commonly assessed in ecotoxicological studies [16]. Studies on L. minor have shown that it contributes to ecotoxicological research as a representative of aquatic plants, and standardized protocols for performing growth inhibition tests on this plant species have been developed [12,17]. It facilitates ecotoxicity tests under controlled laboratory conditions and provides measurable endpoints like frond morphology, biomass, and photosynthetic pigments such as chlorophyll and carotenoid content, making it ideal for assessing the toxicity of environmental pollutants on aquatic ecosystems [12,15].
Due to the adverse ecological and human health effects, TWP derivatives like 4-ADPA are recognized as emerging environmental contaminants [2]. Despite being a pollutant of rising concern, studies on 4-ADPA and its ecotoxicity are limited. Therefore, assessing the ecotoxicological impact of 4-ADPA is important for environmental pollution monitoring and for characterizing the risks to various aquatic life forms, as TWPs are a persistent source of pollution. Among the potentially exposed aquatic species are aquatic macrophytes like Lemna. Additionally, L. minor serves as a suitable model for assessing the toxicity of environmental pollutants due to its relatively small size, rapid growth, and ease of clonal propagation [18].
Considering the above, the present study aimed to observe how the tire-wear-derived compound 4-ADPA influences the growth and photosynthesis of the aquatic macrophyte L. minor and evaluate its ecotoxicological impact on this species.

2. Materials and Methods

2.1. Chemicals and Reagents

The chemical standard of 4-ADPA was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Dimethyl sulfoxide (DMSO) and electronic grade methanol were purchased from Fisher Scientific (Waltham, MA, USA). The basal salt mixture used to prepare Hoagland media was obtained from Caisson Laboratories (Smithfield, UT, USA). An Andonstar AD246SP HDMI digital microscope (Shenzhen Andonstar Tech Co., Ltd., Shenzhen, China) was used for imaging, and ImageJ software (version 1.54p) was used to count the frond number. The Ohaus AV812 Adventurer Pro digital balance (OHAUS Corporation, Parsippany, NJ, USA) was used for measuring plant biomass. A Thermo Scientific Spectronic 200 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify photosynthetic pigments.

2.2. Plant Materials and Culture Conditions

L. minor was collected initially from the Louisiana State University Lake, Baton Rouge, Louisiana, and the duckweed culture was established at the Environmental Health Sciences Laboratory at Southern University and A&M College, Baton Rouge, Louisiana. The plants were grown and maintained in 100 mm × 15 mm Petri dishes with approximately 50 mL of Hoagland’s culture medium. Cultures were maintained under controlled conditions with continuous light exposure (24 h light: 0 h dark photoperiod) and a consistent temperature of 24 ± 2 °C, consistent with OECD guideline 221 [17]. Light intensity within the culture vessels was maintained at a photosynthetic photon flux density (PPFD) of 300 µmol/m2/s. The total medium was renewed once a week.

2.3. Experiment Design

4-ADPA test solutions were prepared by dissolving 4-ADPA crystals in 100% DMSO, followed by dilution in Hoagland’s culture medium to achieve desired concentrations, resulting in a final 1% (v/v) DMSO concentration. The tested 4-ADPA concentrations were 10 μg/L, 25 μg/L, 50 μg/L, and 100 μg/L. Hoagland’s medium served as the negative control.
To address potential solvent effects, a vehicle control group containing 1% DMSO in Hoagland’s medium was included in preliminary experiments. These preliminary tests with L. minor using a range of reported environmentally relevant concentrations of 4-ADPA allowed for the identification of a correlation between growth parameters and photosynthetic pigments, and optimization of the experimental range to effectively evaluate the desired biological response. Statistical analysis of the preliminary data revealed no significant difference in growth parameters between the 1% DMSO vehicle control group and the Hoagland’s medium negative control group. This finding aligns with established research indicating that 1% DMSO can be considered a ‘no observed effect concentration’ (NOEC) for L. minor growth based on endpoints like frond number and area [19]. Consequently, the data from the vehicle control group were excluded from the subsequent statistical analyses comparing the effects of 4-ADPA treatments to the negative control. This approach ensured that any observed effects are attributed to the presence of 4-ADPA and not the vehicle solvent.
Healthy duckweed plants with 2–3 fronds from laboratory stock culture were selected as test specimens. Three Lemna plants with three fronds were exposed in a 6-well culture plate in three replicates with a final volume of 5 mL of Hoagland’s medium per well supplemented with a tested range of 4-ADPA concentrations. The test was conducted for 7-day and 14-day exposure periods under controlled temperature and continuous light exposure, based on OECD guideline 221 [17]. At the end of the 7-day and 14-day experimental periods, fronds from each treatment group were collected, and relative growth rates based on fresh weight and frond number were recorded. Then, the plants were placed in Eppendorf microtubes (Thermo Fisher Scientific, Waltham, MA, USA) for further analysis.

2.4. Plant Growth Determination

The growth rate of L. minor was determined after 7 and 14 days of exposure to different concentrations of 4-ADPA under laboratory conditions. The Lemna fronds were surface-dried on absorbent paper and then weighed in an analytical balance to determine fresh weight. The fronds were photographed using an HDMI digital microscope (Shenzhen Andonstar Tech Co., Ltd., Shenzhen, China), and the frond numbers were counted using ImageJ software (version 1.54p).

2.5. Physiological Markers Analysis

2.5.1. Photosynthetic Pigment Contents

Pigments were extracted from L. minor fronds using 1 mL of 100% methanol per replicate, under room temperature and dark conditions, based on the protocol by Hiscox and Israelstam (1979) [20]. After 24 h, the absorbance of supernatants was measured at 470 nm, 652 nm, and 665 nm using a Thermo Scientific Spectronic 200 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The extraction solution was used as a blank. Photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) were calculated following the equations (Table 1) proposed by Lichtenthaler (1987), and results were expressed in milligrams per gram of fresh weight (mg/g) [21].

2.5.2. Histological Analysis for Starch Accumulation

Starch accumulation was determined following the protocol by Bourgeade et al. (2021) [22]. The decolorized plant tissue after methanolic extraction was gently washed with water, dried, and then stained with Lugol’s solution. Relevant areas of starch accumulation were identified and recorded using an HDMI camera (Shenzhen Andonstar Tech Co., Ltd., Shenzhen, China) coupled to a light microscope.

2.6. Statistical Analysis

All values are presented as the mean ± standard deviation (SD). Before conducting ANOVA, the assumptions of normality and homogeneity of variances were assessed. Normality was tested using the Shapiro–Wilk test, and homogeneity of variances was tested using Levene’s test. The data for all parameters met the assumptions required for analysis of variance.
A one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc t-test was performed to analyze the significant differences between the treatment and control groups. A p-value of less than 0.05 (p < 0.05) was considered statistically significant. SPSS (version 30) was used for all statistical analyses, and Microsoft Excel was used for graphical presentation.
While the sample size (n = 3 replicates per treatment group in each of the three independent experiments, totaling n = 9) was appropriate for initial assessment, it is acknowledged that this sample size may limit the statistical power to detect subtle effects, particularly at lower concentrations of 4-ADPA.

3. Results

We obtained insights into how tire-wear compounds affect aquatic plants’ growth dynamics and physiological response using a laboratory-scale experiment and a simple macrophyte model. This study aimed to assess how the environmental residue of TWPs like 4-ADPA affects aquatic plants and the aquatic ecosystem when washed down the drain.

3.1. Effects of 4-ADPA on Plant Morphology

Post-4-ADPA exposure images obtained with an HDMI microscope show the morphological changes in L. minor (Figure 1). After 7 days of exposure, the Lemna fronds exposed to higher concentrations of 4-ADPA started to exhibit signs of stunted growth and chlorosis, with small, round, and pale green or yellow fronds. A visible gradual growth inhibition was observed with increased concentration of 4-ADPA. A similar trend was observed in the 14-day experiment.

3.2. Effects of 4-ADPA on Growth Parameters

Duckweed samples exhibited signs of stunted growth after 7 days of exposure to 4-ADPA (Figure 1). Consistent findings were evident in growth parameters such as frond number and fresh weight (Figure 2). A statistically significant decrease in frond number was observed at concentrations of 50 μg/L and 100 μg/L of 4-ADPA on day 7 and day 14 of treatment. There was approximately an 80% reduction in fronds after 14 days of exposure to a 100 μg/L concentration of 4-ADPA compared to the control group (Figure 2C). L. minor’s exposure to 4-ADPA led to a dose-dependent reduction in fresh weight after 7 and 14 days of treatment (Figure 2B,D). Fresh biomass significantly decreased after 7 days of exposure to 25 μg/L, 50 μg/L, and 100 μg/L of 4-ADPA compared to the control group (Figure 2B). However, fresh weight increased under exposure to the 10 and 25 μg/L concentrations by day 14 compared to day 7, possibly indicating partial recovery or adaptation of the plants after longer exposure time. Despite this, the progressive decline in fresh weight remained evident among the treatment groups on day 14, and a significant reduction was noted at the 100 μg/L concentration of 4-ADPA (Figure 2D).

3.3. Effects of 4-ADPA on Photosynthetic Pigment Contents

L. minor began to show signs of chlorosis after 7 days of exposure to 4-ADPA (Figure 1). Consequently, there was a marked decrease in chlorophyll a, b, and total chlorophyll contents compared to the control group (Figure 3). L. minor grown under exposure to 10 μg/L, 25 μg/L, 50 μg/L, and 100 μg/L concentrations for 7 days demonstrated a significant dose-dependent reduction in chlorophyll a (Figure 3A). A similar significant result was observed after 14 days of exposure to 25 μg/L, 50 μg/L, and 100 μg/L of 4-ADPA (Figure 3E). Likewise, a time- and dose-dependent decline in chlorophyll b was noted in the exposed plants, with a greater decline observed on day 14 with 100 μg/L of 4-ADPA (Figure 3F). After 7 days of exposure, a progressive decline in chlorophyll b was recorded, with significant results at 25 μg/L, 50 μg/L, and 100 μg/L of 4-ADPA (Figure 3B), while similar effects were noted at 50 μg/L and 100 μg/L of 4-ADPA after 14 days (Figure 3F). The time and dose-dependent inhibitory effect of 4-ADPA exposure was also evident in the total chlorophyll content. After 7 and 14 days of exposure, there was approximately a 50% reduction in total chlorophyll content at 100 μg/L of 4-ADPA, and a statistically significant reduction was observed even at the lower concentration of 10 μg/L of 4-ADPA after 7 days of exposure. Additionally, a significant reduction in total chlorophyll was observed at 10 μg/L, 25 μg/L, 50 μg/L, and 100 μg/L for 7 days and at 50 μg/L and 100 μg/L for 14 days (Figure 3C,G). Comparably, no significant effects were observed in carotenoid content after 7 days of 4-ADPA exposure (Figure 3D). However, a dose-dependent declining trend was observed after 14 days of exposure, and significance was observed at 100 μg/L of 4-ADPA (Figure 3H).

3.4. Histological Evaluation of Starch Accumulation

The iodine in Lugol’s stain binds to the starch molecules, producing a dark coloration [23]. Staining with Lugol’s solution and microscopic analysis demonstrated increased areas of starch accumulation in L. minor with a higher concentration of 4-ADPA treatment (Figure 4). A 14-day exposure yielded similar results. Despite decreased photosynthetic pigments with increased 4-ADPA exposure, increased starch accumulation can be linked to plant stress conditions.

4. Discussion

Lemna minor, a common duckweed, has been widely used as a study model in ecotoxicology to assess how toxic some chemicals or pollutants might be to non-target species because of its high sensitivity to a wide range of chemicals, short reproduction cycle, and ease of growth, maintenance, and manipulation in the laboratory [23]. In addition, duckweeds, an integral part of the aquatic ecosystem that grow in aggregates of colonies forming large blankets across the water surface, play a key role in habitat provision, phytoremediation, and nutrient cycling, so they are also an ecologically significant species [12,24]. In recent years, the toxicity and hidden threats of tire wear leachates to aquatic habitats and species like coho salmon, rainbow trout, algae, L. minor, etc., have been studied widely [25,26,27,28]. Various types of TWPs are generated by the friction between the tire and the road surface, accounting for more than 50% of microplastics in the environment [29]. While the concentrations of 4-ADPA in aquatic systems of interest may not be readily available in the published literature, it is a degradation product of the tire rubber chemical 6-PPD and can enter aquatic systems through processes like storm water runoff. Studies have shown that 4-ADPA can be detected in urban creek water and wastewater treatment plant effluent, indicating its presence in aquatic environments and highlighting the importance of evaluating its ecotoxicological impact [2]. Despite 4-ADPA being one of the tire-derived pollutants, its effects on the aquatic environment and species remain poorly understood.
This study tested tire-derived compound 4-ADPA for its aquatic ecotoxicity using L. minor as a study model. The 4-ADPA treatment suppressed the growth of plants, with visible chlorosis and disintegrating fronds in the exposed L. minor plants (Figure 1). The number of fronds also substantially decreased in a dose-dependent manner following 4-ADPA exposure (Figure 2A). Fresh biomass was reduced by more than 50% after 7 days of exposure (Figure 2B). By day 14, fresh weight increased compared to day 7 of the experiment, suggesting the plant’s potential adaptation and tolerance over time; however, a dose-dependent reduction was still evident across the treatment groups [30] (Figure 2B). The growth-related parameters of this study align with findings reported by Kumar et al., where chlorosis and a significant decrease in growth based on frond number and dry weight were observed following exposure to TWP leachates [31]. Chlorosis is the progression from green-colored fronds to pale, yellow fronds. It is attributed to reduced chloroplast density per cell and chlorophyll content due to nutrient deficiency, oxidative stress, bacterial or fungal infestations, chemical exposure, pollution, etc. [24]. Chlorosis affects photosynthetic efficacy, leading to a decreased ability of plants to synthesize carbohydrates and produce energy. This can result in stunted growth, detachment of some fronds, and plant death [24].
Not only was the plant’s growth affected after 4-ADPA treatment, but a similar pattern was observed with the photosynthetic pigment’s analysis. The decline in growth-related parameters correlated closely with the physiological biomarkers like chlorophyll and carotenoid contents in L. minor exposed to 4-ADPA. In our study, a dose-dependent reduction in pigments like chlorophyll a, chlorophyll b, and total chlorophyll was observed (Figure 3). A similar outcome was also observed in the ecotoxicological studies by Radic et al. [30] and Kumar et al. [31], which supports our findings. Chlorophyll is a pigment within the plant’s chloroplasts, giving plants a characteristic green color. The amount of chlorophyll in leaf tissue is affected by nutrient deficiency and environmental conditions like pollutants, temperature, drought, etc. Plants have two types of chlorophyll pigment, chlorophyll a and chlorophyll b, which play a unique role in the physiology and productivity of green plants. Therefore, estimating chlorophyll contents has been of special interest in assessing physiological processes and plant health. Chlorophyll a is a bluish-green pigment and a primary pigment that converts light energy into chemical energy during photosynthesis. It is about three times more abundant than chlorophyll b in plant tissue. Chlorophyll b is an accessory, yellowish green pigment that helps to absorb light energy at different wavelengths and transfer it to chlorophyll a. Total chlorophyll is simply the sum of chlorophyll a and chlorophyll b [32,33]. Carotenoids are essential pigments of terpenoid groups crucial to photosynthesis, phytoprotection, and structural stabilization of photosynthetic complexes [24,34]. The carotenoid content was not significantly affected after 7 days of 4-ADPA exposure. However, after 14 days, the carotenoid content progressively declined with increased 4-ADPA exposure, particularly at higher concentrations. This delayed effect suggests that L. minor may have initial compensatory or protective mechanisms to maintain carotenoid levels under moderate or short-term exposure to 4-ADPA. The decline observed after 14 days could be indicative of prolonged stress overwhelming these initial defenses, leading to a breakdown of carotenoid synthesis or an increase in degradation [35]. In our study, the effects of 4-ADPA on carotenoid content were less apparent compared to those on chlorophylls, consistent with the findings by Radic et al. [30]
The Lugol staining revealed increased starch accumulation with increased 4-ADPA concentration, which aligns with the results reported by Sree et al. [36]. In this method, the iodine in Lugol’s solution reacts with starch, making the intracellular starch visible and producing a dark coloration [23]. Starch forms a helical structure of its monomer glucose, and the iodine from Lugol’s solution becomes trapped in the glucose helix, thus producing purple-black-colored areas [37]. The decreased chlorophyll content with increased 4-ADPA treatment reflected impaired photosynthesis and decreased carbohydrate synthesis. However, increased starch accumulation with higher concentrations of 4-ADPA was detected with Lugol’s staining. This is attributed to the stress the plants are under. Environmental conditions, like pollutants, can cause stressed conditions in plants, leading to less carbohydrate being used for growth, with the excess being stored as starch [36,38].
This apparent contradiction between reduced photosynthesis and increased starch accumulation suggests a disruption in the balance between carbohydrate synthesis, utilization, and storage under 4-ADPA stress. Stress conditions can affect starch metabolism by impacting photosynthetic rates and altering carbon allocation [39]. While starch degradation often provides energy during abiotic stress, accumulation can occur if growth inhibition is more pronounced than the effect on photosynthesis [40]. Additionally, high starch accumulation can lead to feedback inhibition of photosynthesis, potentially through reduced carbon sink strength [41]. The observed decrease in chlorophyll content might be a consequence of this feedback. Further research into how 4-ADPA affects carbohydrate metabolism, potentially through altered enzyme activities involved in starch synthesis or degradation or altered signaling pathways that regulate carbon allocation, is needed to fully understand these responses [40].
This study adds to our understanding of how TWPs disrupt the aquatic ecosystem. The observed chlorosis with stunted growth, reduced photosynthetic pigments, and increased starch accumulation with exposure to increased concentrations of 4-ADPA negatively affect photosynthetic efficiency in plants and can be linked to various stress conditions [24,38]. The reduced growth and physiological biomarkers with increased 4-ADPA treatment demonstrate the ecotoxicological effects of 4-ADPA on L. minor; however, our study focuses on only one species, which may not represent the effects on other marine habitats. Therefore, future studies with additional model species like zebrafish, coho salmon, Caenorhabditis elegans, etc., are suggested for a broader understanding of the ecological impact of 4-ADPA. To clarify the mechanisms of 4-ADPA toxicity, future studies should investigate whether it causes oxidative stress or interferes with photosynthetic enzyme activity.

5. Conclusions

To our knowledge, no previous studies have investigated the ecotoxicological impacts of the compound 4-ADPA on L. minor. The observed morphological changes, along with data from the growth parameters (frond number and fresh weight) and physiological responses, demonstrated comparable patterns, indicating that higher concentrations of 4-ADPA can have a more pronounced effect, reducing L. minor’s ability to grow and reproduce and impacting its ecological significance in the aquatic environment. While L. minor is a valuable model species for initial assessments, these findings highlight the necessity for future research involving additional aquatic species (e.g., algae, fish) to comprehensively assess 4-ADPA’s broader ecological impacts.

Author Contributions

E.H. conceived, designed and supervised the research; S.K. and N.C. performed experiments; E.H. and S.K. analyzed data; E.H. and S.K. interpreted the experimental results; E.H. and S.K. prepared figures; E.H. and S.K. drafted the manuscript; A.A., K.M., N.C., R.R., S.K. and E.H. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was partially supported by the National Science Foundation (NSF) through the Historically Black Colleges and Universities—Excellence in Research (HBCU-EiR) program (Grant number: 2200607) and by Southern University Research and Enhancement Development (RED) grant (Grant number REDG2122-RR) awarded to R.R. (P.I.) and E.E. (Co-P.I.). Additionally, E.H.’s laboratory received support from a pilot research award from Louisiana Biomedical Research Network (LBRN), which is funded by the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under grant number P20 GM103424-21. The content of this manuscript is solely the responsibility of the authors and does not necessarily reflect the official views of the funding agencies.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to thank Masomeh Fatemi and Mary Beals for their invaluable technical assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
TWPsTire Wear Particles
4-ADPA4-Aminodiphenylamine
DMSODimethyl Sulfoxide
OECDOrganisation for Economic Co-operation and Development
ANOVAAnalysis of Variance

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Pollutants 05 00020 g001
Figure 1. The morphology of L. minor treated with different concentrations of 4-ADPA for 7 days. Images were taken using an HDMI digital microscope.
Figure 1. The morphology of L. minor treated with different concentrations of 4-ADPA for 7 days. Images were taken using an HDMI digital microscope.
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Figure 2. Growth-related effects of 4-ADPA on L. minor after 7 and 14 days of exposure. L. minor plants were exposed to 4-ADPA at concentrations of 10, 25, 50, or 100 μg/L, or kept in a control group without 4-ADPA. The figures show frond number and fresh weight after 7 days (A,B) and 14 days (C,D) of exposure. The study involved three independent experiments with three technical replicates each, resulting in n = 9 replicates per treatment group. Growth parameters are presented as the mean ± SD from the pooled data (n = 9). Significant differences compared to the control are indicated by an asterisk (* p < 0.05, ** p < 0.001).
Figure 2. Growth-related effects of 4-ADPA on L. minor after 7 and 14 days of exposure. L. minor plants were exposed to 4-ADPA at concentrations of 10, 25, 50, or 100 μg/L, or kept in a control group without 4-ADPA. The figures show frond number and fresh weight after 7 days (A,B) and 14 days (C,D) of exposure. The study involved three independent experiments with three technical replicates each, resulting in n = 9 replicates per treatment group. Growth parameters are presented as the mean ± SD from the pooled data (n = 9). Significant differences compared to the control are indicated by an asterisk (* p < 0.05, ** p < 0.001).
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Figure 3. Physiological effects of 4-ADPA on L. minor after 7 and 14 days of exposure. L. minor plants were exposed to 4-ADPA at concentrations of 10, 25, 50, or 100 μg/L, or kept in a control group without 4-ADPA. Photosynthetic pigment concentrations are shown after 7 days ((A–D): chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) and 14 days ((E–H): chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids). Pigment concentrations are expressed in milligrams per gram of fresh weight (mg/g). The study involved three independent experiments with three technical replicates each, resulting in n = 9 replicates per treatment group. Data represent the mean ± SD from the pooled data (n = 9). Significant differences compared to the control are indicated by an asterisk (* p < 0.05, ** p < 0.001).
Figure 3. Physiological effects of 4-ADPA on L. minor after 7 and 14 days of exposure. L. minor plants were exposed to 4-ADPA at concentrations of 10, 25, 50, or 100 μg/L, or kept in a control group without 4-ADPA. Photosynthetic pigment concentrations are shown after 7 days ((A–D): chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) and 14 days ((E–H): chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids). Pigment concentrations are expressed in milligrams per gram of fresh weight (mg/g). The study involved three independent experiments with three technical replicates each, resulting in n = 9 replicates per treatment group. Data represent the mean ± SD from the pooled data (n = 9). Significant differences compared to the control are indicated by an asterisk (* p < 0.05, ** p < 0.001).
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Figure 4. Effects of 4-ADPA on starch accumulation in L. minor. L. minor plants were exposed to the control and 4-ADPA treatments (10 μg/L, 25 μg/L, 50 μg/L, 100 μg/L). Microscopic analysis of the starch accumulation by Lugol’s staining of L. minor fronds after 7 days of exposure to different concentrations of 4-ADPA. Images were taken using an HDMI digital microscope.
Figure 4. Effects of 4-ADPA on starch accumulation in L. minor. L. minor plants were exposed to the control and 4-ADPA treatments (10 μg/L, 25 μg/L, 50 μg/L, 100 μg/L). Microscopic analysis of the starch accumulation by Lugol’s staining of L. minor fronds after 7 days of exposure to different concentrations of 4-ADPA. Images were taken using an HDMI digital microscope.
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Table 1. Equations for the determination of chlorophyll a (Ca), chlorophyll b (Cb), Total chlorophyll (Ca+b), and carotenoids in Lemna minor extracts using methanol as a solvent. The pigment concentrations were obtained by inserting the respective absorbance values in the given equation.
Table 1. Equations for the determination of chlorophyll a (Ca), chlorophyll b (Cb), Total chlorophyll (Ca+b), and carotenoids in Lemna minor extracts using methanol as a solvent. The pigment concentrations were obtained by inserting the respective absorbance values in the given equation.
PigmentEquation
Ca(16.72 ∗ A665.2) − (9.16 ∗ A652.4)
Cb(34.09 ∗ A652.4) − (15.28 ∗ A665.2)
Ca+b(1.44 ∗ A665.2) − (24.93 ∗ A652.4)
Carotenoids(1000 ∗ A470 − 1.63 ∗ Ca − 104.96 ∗ Cb)/221
A is the absorbance in 1 cm cuvette.
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MDPI and ACS Style

Kandel, S.; Campbell, N.; Abdulkadir, A.; Moore, K.; Rosby, R.; Hossain, E. Assessment of Growth and Physiological Responses of Lemna minor Exposed to 4-Aminodiphenylamine, a Tire Wear Compound. Pollutants 2025, 5, 20. https://doi.org/10.3390/pollutants5030020

AMA Style

Kandel S, Campbell N, Abdulkadir A, Moore K, Rosby R, Hossain E. Assessment of Growth and Physiological Responses of Lemna minor Exposed to 4-Aminodiphenylamine, a Tire Wear Compound. Pollutants. 2025; 5(3):20. https://doi.org/10.3390/pollutants5030020

Chicago/Turabian Style

Kandel, Shila, Naja’Ree Campbell, Abubakar Abdulkadir, Kristin Moore, Raphyel Rosby, and Ekhtear Hossain. 2025. "Assessment of Growth and Physiological Responses of Lemna minor Exposed to 4-Aminodiphenylamine, a Tire Wear Compound" Pollutants 5, no. 3: 20. https://doi.org/10.3390/pollutants5030020

APA Style

Kandel, S., Campbell, N., Abdulkadir, A., Moore, K., Rosby, R., & Hossain, E. (2025). Assessment of Growth and Physiological Responses of Lemna minor Exposed to 4-Aminodiphenylamine, a Tire Wear Compound. Pollutants, 5(3), 20. https://doi.org/10.3390/pollutants5030020

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