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Frontiers in Ecology and Evolution ·

Volume 14 - 2026 | https://doi.org/10.3389/fevo.2026.1938255

Effect of diesel oil water-soluble fraction on physiological characteristics of Thalassia hemprichii and its integrated biomarker response

  • Siyang Ma1,2
  • Muqiu Zhao1,2,3
  • Kang Wang1,2
  • Zhen Ni1,2,3*corresponding author
  • Yunfeng Shi1,2,4*corresponding author
  • Yuge Li1,4
  • Yupeng Feng1
  • Qiuying Han1,3
  1. 1Yazhou Bay Innovation Institute of Hainan Tropical Ocean University, Hainan Tropical Ocean University, Sanya, China
  2. 2Key Laboratory for Coastal Marine Eco‑Environment Process and Carbon Sink of Hainan Province, Hainan Tropical Ocean University, Sanya, China
  3. 3Key Laboratory of Utilization and Conservation for Tropical Marine Bioresources, Ministry of Education, Sanya, China
  4. 4Hainan Provincial Modern Marine Ranching Engineering Research Center, Hainan Tropical Ocean University, Sanya, China
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Abstract

Petroleum and its product pollution is a leading cause of seagrass meadow degradation. However, the dose–response relationship between diesel water-soluble fraction (DWSF) and seagrass physiological characteristics remains poorly understood. This study aimed to elucidate the physiological response mechanisms of a typical tropical seagrass to DWSF stress. The tropical dominant seagrass Thalassia hemprichii was exposed to different concentrations of 0# DWSF for 15 days in a semi-static water simulation. Leaf growth rate, photosynthetic physiological parameters, and oxidative stress biomarkers were measured. The integrated biomarker response (IBR) method was applied to classify and quantify the physiological responses of Thalassia hemprichii to DWSF. DWSF induced significant concentration-dependent physiological effects. Leaf growth rate was significantly inhibited when DWSF exceeded 1/30 of the saturation concentration (P<0.05). Respiration rate, chlorophyll b content, Fv/Fm, ETR, qP, and ΦPSII all decreased with increasing DWSF concentration. Net photosynthetic rate, chlorophyll a content, and NPQ showed a unimodal trend (increase then decrease), with the inflection point at 1/100–1/30 saturation concentration. Antioxidant enzyme activities (POD, SOD, CAT, APX) and oxidative stress products (MDA, H2O2) increased with DWSF concentration. IBR values based on photosynthetic pigment or oxidative stress parameters were highly significantly positively correlated with DWSF concentration (P<0.01). Physiological stress on Thalassia hemprichii intensified with increasing DWSF concentration, manifesting as growth inhibition, photosynthetic impairment, and antioxidant defense activation. Nevertheless, Thalassia hemprichii exhibited strong tolerance to DWSF within the tested concentration range. The IBR method can effectively evaluate the impact of DWSF on the physiological characteristics of seagrass.

Keywords

1. Introduction

Seagrass is the only group of higher angiosperms that grows entirely in seawater on Earth (Duffy, 2006). In the intertidal or subtidal zones, ecosystems formed by one or more seagrass species are known as seagrass meadows (Hemminga and Duarte, 2000). With high primary productivity and a strong capacity to capture suspended solids, the seagrass meadow is not only a vital ‘blue carbon’ ecosystem but also plays a crucial role in purifying water bodies, stabilizing sediments, providing food and spawning grounds for various marine organisms. It possesses extremely important ecological functions (Zhou et al., 2016). In recent years, affected by climate change and human activities, global seagrass meadows are declining at a rate of 7% per year, and over 80% of the seagrass meadows in China’s coastal waters have disappeared (Orth et al., 2006; Zhou et al., 2023).

Diesel oil is one of the most widely used light petroleum products in China, and it is also the most commonly used fuel type for offshore power and power acquisition. The pollution caused by its leakage is one of the most common types of pollution in China ‘s coastal waters. Diesel oil is mainly composed of alkanes, aromatic hydrocarbons, polycyclic aromatic hydrocarbons and other non-hydrocarbon substances. Once the leakage of diesel oil occurs at sea, its components will gradually spread under the action of wind, waves, currents and tides, and exist in various forms such as floating oil film, dissolved state, emulsified state and condensed state, causing large-scale marine pollution and threatening marine life and ecology (Voskoboinikov et al., 2020). It is generally believed that the components dissolved or emulsified in water are the forms of direct toxicity of petroleum products to aquatic organisms (Lv and Xiong, 2010). The toxic effects of oil intake by marine organisms are usually determined by the composition and absolute content of small and medium molecular weight alkanes (C 12 - C 24) and polycyclic aromatic hydrocarbons (such as naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, etc.). Since the water solubility of aromatics is higher than that of saturated hydrocarbons, the effective toxic components contained in diesel oil water-soluble fraction (DWSF) are much higher than those in refined oil (Sun et al., 2018; Singh et al., 2020). In view of the fact that the impact of marine oil spill on the ecological environment has become a hot issue of concern, the effects of DWSF on marine plankton, benthic animals and fish have been widely reported (Yu et al., 2005; Simonato et al., 2008; Santos et al., 2010; Dong et al., 2016; Sun et al., 2019), but the research on its physiological and ecological effects on tropical seagrasses is very limited. Nevertheless, most published petroleum-toxicity studies on seagrasses have focused on crude oil or chemically dispersed oil, predominantly examining temperate Zostera species. These works have demonstrated that petroleum contaminants can impair photosynthetic function, reduce pigment concentration and inhibit seagrass growth. In contrast, experimental data regarding the biological consequences of diesel water-soluble fraction exposure on tropical seagrass species are still insufficient (Fonseca et al., 2017).

Biomarkers refer to the changes in the growth, physiology or behavior of organisms in response to one or more stresses at the body fluid, tissue or individual level (Boudjme et al., 2023). Since there is a certain quantitative relationship between this response change and environmental stress, the application of biomarkers can effectively predict the content level, biological effects and toxicological characteristics of pollutants in the environment. The morphological, physiological, pathological and histological parameters of organisms can be used as biomarkers, among which oxidative stress-related parameters are the most commonly used (van der Oost et al., 2003; Li et al., 2021). However, the sensitivity of different biomarkers to environmental stress is different and the response is not synchronized, so it is limited to use a single biomarker to evaluate the stress effect. The integrated biomarker response (IBR) index can quantitatively analyze the impact of environmental pollutants on organisms by comprehensively analyzing the response of multiple biomarkers to environmental stress and converting it into a ‘ pressure index ‘ value (Lin et al., 2016; van der Oost et al., 2003; Marigómez et al., 2013). It has been widely used in the monitoring and ecological effect evaluation of marine pollutants (Pan et al., 2022; Liu et al., 2024; Zhou et al., 2024). For example, Cao et al. (2023) applied IBR to evaluate the pollution status of marine PAHs in Laizhou Bay; Chi et al. (2020) studied the physiological effects of polybrominated diphenyl ether flame retardants on Cynoglossus semilaevis based on IBR model. Brooks et al. (2012) and Pytharopoulou et al. (2008) studied the response of IBR index of Mytilus edulis and Mytilus galloprovincialis to sewage discharge and heavy metal pollution, respectively. Zhang et al. (2015) and Jiang et al. (2015) evaluated the toxic effects of DWSF on Ruditapes philippinarum and Sinonovacula constricta using the IBR index, respectively. However, up to now, the application of IBR to comprehensively evaluate the effects of DWSF on the physiological characteristics of tropical seagrass has not been reported.

Thalassia hemprichii is one of the dominant seagrass species in the tropical western Pacific and Indian Ocean. It is mainly distributed in the southern coast of Taiwan, Hainan Island and the surrounding waters of the South China Sea islands and reefs in China (Cai et al., 2017). In this study, T. hemprichii was used as the test organism, and the effects of different concentrations of DWSF on the growth rate, dark respiration and net photosynthetic rate, photosynthetic pigment content, photosynthetic fluorescence parameters, antioxidant enzyme activity and oxidative stress product content of seagrass leaves were explored through semi-hydrostatic simulation experiments. At the same time, the photosynthetic rate, photosynthetic fluorescence, photosynthetic pigments and oxidative stress were used to construct the IBR system to evaluate the physiological response of T. hemprichii after exposure to DWSF, and the IBR model with high sensitivity and good linear relationship was screened. The results of this study can effectively reveal the physiological and ecological effects of diesel pollution on seagrass, provide technical support for the monitoring and evaluation of the ecological health of seagrass meadows, and provide a basis for the restoration and management of degraded seagrass meadows.

2. Materials and methods

Acquisition and domestication of seagrass

In this study, Thalassia hemprichii was collected from the seagrass meadow of Xincun Bay, Lingshui County, Hainan Province (18°24′46″N, 109°59′30″E) during the ebb tide in November 2023. Seagrass ramets were excavated intact with approximately 20 cm of underlying sediment to preserve complete underground rhizome structures. Attached surface sediments and epibionts were gently rinsed with local seawater, and samples were immediately transported to the laboratory in seawater-filled insulated containers.

Uniform and healthy seagrass ramets with consistent morphological status were selected for experimentation. Each standardized individual contained one intact shoot and a 10 cm-length rhizome segment. Prior to DWSF exposure, all seagrass materials underwent a 7-day hydroponic acclimation period in glass containers to adapt to laboratory conditions. During acclimation, the seawater temperature was maintained at 27°C with a salinity of 32 ‰. A circulating pump was used to maintain continuous water flow, and the culture seawater was completely renewed every 24 h to ensure water quality stability. Background environmental parameters of the sampling site in the corresponding season, summarized from published regional investigations, are provided in Supplementary Material.

Preparation of DWSF saturated mother liquor

The diesel used in this study is commercially available 0 # diesel. After the diesel oil was transported back to the laboratory with a sealed metal barrel, it was mixed with seawater (salinity 32 ‰) filtered by a 0.45 μm filter membrane at a ratio of 1: 10 and placed in a stirrer for continuous high-speed stirring for 12 h. After stirring, the lower solution was taken as the saturated mother liquor of DWSF after sufficient standing (≥ 24 h) and stored in a brown bottle at low temperature for later use (Jia and Lin, 1998). The concentration of DWSF (petroleum hydrocarbon) in the mother liquor was determined by ultraviolet spectrophotometry (Chen, 2020), which was 83.90 mg/L.

DWSF pollution stress test

In order to avoid the adsorption of dissolved petroleum hydrocarbons in seawater by the substrate and the large concentration change of DWSF due to degradation and volatilization during the culture process, this study used indoor a semi-static water simulation study, and no substrate was used during the culture process. The rhizomes of 10 seagrass standard plants were wrapped in clean, thin metal wires on clean glass rods and placed in an integrated glass incubator (length × width × height = 30 cm × 18 cm × 20 cm). The seawater containing DWSF (salinity 32 ‰) 8.1 L (15 cm deep) submerged the seagrass leaves.

A total of 6 concentrations were set up in the experiment, namely: CK (control, without DWSF), BH/100 (DWSF concentration was 1/100 of saturated concentration, that is, 0.84 mg/L). Slightly higher than the ‘ Seawater Quality Standard ‘ (GB 3097-1997), BH/30 (DWSF concentration is 1/30 of the saturated concentration, that is 2.80 mg/L), BH/10 (DWSF concentration is 1/10 of the saturated concentration, that is 8.39 mg/L), BH/3 (DWSF concentration is 1/3 of the saturated concentration, that is 27.97 mg/L), BH (DWSF concentration is saturated concentration, that is 83.90 mg/L). Cultured seawater with different DWSF concentrations was prepared by diluting the mother liquor with seawater, and three replicates were set for each treatment.

The lowest DWSF concentration (0.84 mg/ L) slightly exceeded the petroleum-hydrocarbon threshold of Class-IV seawater (0.50 mg /L, GB 3097-1997). Medium exposure concentrations generally cover the range commonly used in laboratory diesel-water-accommodated-fraction toxicity experiments on coastal aquatic plants, simulating transient local high-pollution conditions after in-shore fuel spill incidents. The saturated DWSF treatment was included to examine the upper physiological tolerance of T. hemprichii under acute short-term stress, and this concentration substantially exceeds typical ambient seawater petroleum levels (Liu et al., 2025).

It should be noted that only total petroleum hydrocarbon (TPH) concentration of the DWSF stock solution was measured in the present study, while separate quantitative analysis for individual hydrocarbon fractions (e.g., alkanes, polycyclic aromatic hydrocarbons) was not performed. The DWSF concentration gradient was designed to cover environmentally relevant TPH levels in coastal seawater, including natural background concentrations and moderately-elevated pollution scenarios that may arise from near-shore diesel spills. All exposure solutions were serially diluted from one identical saturated DWSF stock to ensure consistent component proportions across treatments.

During the cultivation process, the indoor temperature was controlled at 27 °C; the photosynthetically active radiation (PAR) on the surface of seawater in the incubator was set to 200 μmol/(m 2 ·s, and the daily illumination time was 12 h. Fresh test seawater with the same DWSF concentration was replaced every 48 h. Because the leaves of T. hemprichii are replaced about once every half a month, the culture time was set to 15 days in this study. All physiological parameters were measured after the culture.

Determination of growth and physiological parameters of Thalassia hemprichii

Growth rate

During the experiment, the growth rate of T. hemprichii was determined by pinhole labeling method (Zieman, 1974).

Dark respiration rate and net photosynthetic rate

RR and NPR of T. hemprichii leaves were measured by liquid phase oxygen electrode (Oxygraph +, Hansatech Instruments). Before the determination of RR, the leaves were pre-cultured in dark conditions for more than 6 h. Before the determination of NPR, the leaves were pre-irradiated for more than 2 h under the condition of PAR of 200 μmol/(m 2 ·s). During the determination, the reaction chamber of the electrode was filled with sterilized seawater and a certain amount of seagrass leaves were added. The changes of dissolved oxygen content in seawater under dark conditions and PAR of 200 μmol/(m 2 ·s) were read respectively. The amount of O 2 consumed or released by fresh seagrass leaves per unit mass (g) per unit time (min) was calculated, and the unit was μmol/(min·g).

Photosynthetic pigment content

The content of photosynthetic pigments in the leaves of T. hemprichii was determined by 80% acetone extraction colorimetry (Shu et al., 2010). In this study, the contents of chlorophyll a (Chla), chlorophyll b (Chlb) and carotenoids (C x) were measured respectively. The results were expressed as mg/g of photosynthetic pigments contained in each g of fresh seagrass leaves, that is, mg/g. The content of Chl (a + b) in leaves was the sum of Chla and Chlb. Chla/Chlb is the ratio of Chla to Chlb. Total photosynthetic pigment content (CT) was the sum of Chla, Chlb and C x content.

Photosynthetic fluorescence parameters

The photosynthetic fluorescence parameters of seagrass leaves were measured by underwater modulated chlorophyll fluorescence spectrometer (DIVING-PAM-II, Germany). Before the determination of the induction curve, the seagrass was placed in dark conditions for dark adaptation for 20 min. The initial fluorescence intensity (F0) and the maximum fluorescence intensity (Fm) were automatically measured by WinControl 3.0 (Walz) software under the conditions of photochemical light intensity of 200 μmol/(m 2 ·s) and saturated pulsed light intensity of 3 000 μmol/(m 2 ·s). The maximum photosynthetic efficiency (F v / F m) of photosystem II (PSII), actual photochemical quantum efficiency of PSII (Φ PSII), apparent photosynthetic electron transport rate (ETR), photochemical quenching coefficient (q P) and non-photochemical quenching coefficient (NPQ) were calculated.

Antioxidant enzyme activity and MDA and H 2 O 2 content

The activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and the contents of MDA and H 2 O 2 in the leaves of T. hemprichii were determined using kits purchased from Nanjing Jiancheng Institute of Bioengineering. The activity of ascorbate peroxidase (APX) was determined using kits purchased from Beijing Solebao Technology Co., Ltd.

Data processing

The processed data were expressed as mean ± standard deviation (SD). Excel 2019 was used for data processing, and SPSS 26.0 was used for one-way ANOVA analysis. Duncan method was used to test the significance of the mean values of each index between different treatments (P < 0.05). Pearson correlation coefficient method was used to analyze the correlation of the data, and GraphPad Prism 8.2.0 was used for plotting.

According to the theory established by Beliaeff and Burgeot (2010) and referring to the method modified by Oliveira et al. (2009), the IBR values of each treatment were calculated. Detailed calculations are given in Equations 1–4.

In the formula, Y is the standardized value of a biomarker, x is the average value of each treatment of the biomarker, m is the total average value of all treatments of the biomarker, and s is the total standard deviation.

In the formula: B i is the length of the radiation line corresponding to the biomarker in each treatment in the star diagram; when biomarkers were induced by pollution stress, Z = Y, otherwise Z =- Y; | Y min | is the absolute value of the minimum Y value of the biomarker.

In the formula, S is the triangle area surrounded by the radiation of adjacent biomarkers, and n is the number of selected biomarkers.

For IBR calculation, the sign parameter Z of each biomarker was predefined according to its physiological function and remained identical across all exposure concentrations, instead of being dynamically adjusted for each treatment. Biomarkers whose suppression represents stress damage (RR, NPR, F v / F m, ETR, q P, Φ PSII) were assigned Z = −Y. NPQ, whose up-regulation indicates activated photoprotective response, was assigned Z = −Y. Even for biomarkers showing biphasic low-stimulation-high-inhibition responses (e.g., NPR), the predefined sign was maintained during calculation. This setting preserves non-monotonic dose-response characteristics but may underestimate the biological stimulation effect at low pollutant concentrations, which should be taken into account when interpreting IBR results. F0 was measured but excluded from the IBR biomarker set.

3. Results

Growth rate

As shown in Figure 1, during the experiment, the growth rate of T. hemprichii leaves treated with CK was (5.5 ± 0.26) mm/d. When the concentration of DWSF in seawater was 1/100 of the saturated concentration (i.e., BH/100 treatment), there was no significant difference in the growth rate of leaves compared with CK (P >0.05). The growth rate of seagrass leaves in each treatment with DWSF concentration ≥ 1/30 saturated concentration was lower than that in BH/100 treatment, and there was no significant difference between the treatments (P >0.05).

RR and NPR

As shown in Figure 2, the RR of T. hemprichii leaves treated with CK was the highest, which was(181 ± 13) μmol/(min·g). The RR of leaves under stress was significantly lower than that of CK (P < 0.05) and decreased with the increase of DWSF concentration. When PAR was 200 μmol/(m 2 ·s), the NPR of T. repens leaves increased first and then decreased with the increase of DWSF concentration. The NPR of BH/30 and BH/100 treatments was 18.62% and 45.41% higher than that of CK treatment, respectively. When the concentration of DWSF reached 1/10 of the saturated concentration (BH/10 treatment), the NPR of leaves decreased significantly compared with BH/30 and then remained at a low level of about 150 μmol/(min·g).

NPR showed a non-monotonic concentration-response pattern and reached its highest value at the intermediate BH/30 treatment. This biphasic trend may be attributable to hormesis-like compensatory regulation: moderate DWSF stress triggered short-term up-regulation of net photosynthesis, whereas pollutant damage overwhelmed self-protective mechanisms above this exposure threshold. Respiratory rate (RR) was generally inhibited in all DWSF treatments, although minor inter-treatment fluctuations were observed. Such non-linear responses are common in aquatic plant toxicology and may also be partially affected by small experimental heterogeneity among replicates.

Photosynthetic pigments

As shown in Figure 3, the Chla content of T. hemprichii leaves showed the phenomenon of low-dose stimulation and high-dose inhibition with the increase of DWSF concentration, and the highest value appeared in BH/100 treatment. Then, with the increase of DWSF concentration, Chla content decreased. The Chla content of seagrass leaves treated with BH was the lowest, which was only 62.34% of CK treatment and 50.26% of BH/100 treatment. The change trend of Chlb content in the leaves of Tailai grass was different from that of Chla, which generally decreased with the increase of DWSF. The Chlb content of seagrass leaves treated with BH was only 39.44% of that of CK treatment. There was no significant difference in C x content in the leaves of T. hemprichii among the treatments (P >0.05). Under the influence of Chla content, the contents of Ch (a + b) and CT in the leaves of T. hemprichii showed the phenomenon of low-dose stimulation and high-dose inhibition with the increase of DWSF concentration. Under the combined effect of Chla and Chlb content, Chla/Chlb did not change much with the increase of DWSF concentration.

Photosynthetic fluorescence parameters

As shown in Table 1, after dark adaptation, the initial fluorescence intensity (F 0) of T. hemprichii leaves increased first and then decreased with the increase of DWSF concentration in seawater, and the inflection point appeared at the DWSF concentration of 1/30 saturated concentration. The maximum fluorescence intensity (F m) decreased with the increase of DWSF concentration. Among all the parameters reflecting the light use efficiency of seagrass leaves, F v / F m, Ф PSII, ETR and q P decreased with the increase of DWSF concentration, and only NPQ increased with the increase of DWSF concentration.

Antioxidant enzyme activity and oxidative stress product content

As shown in Figure 4, the changes of POD, SOD, CAT and APX activities in the leaves of different treatments with the increase of DWSF concentration in seawater were basically the same: when the DWSF concentration was low (≤1/30 saturated concentration), there was no significant difference between the enzyme activities and CK treatment (P >0.05). When the concentration of DWSF exceeded 1/30 of the saturated concentration, the activity of each enzyme began to increase with the increase of DWSF concentration, among which the activity of CAT and APX increased greatly, while the activity of POD and SOD increased slightly. With the increase of DWSF concentration, the contents of oxidative stress products H 2 O 2 and MDA in the leaves of T. hemprichii also increased. The sensitive range of H 2 O 2 content appeared in the range of 1/100-1/10 saturated DWSF concentration, while the sensitive range of MDA content appeared in the range of 0-1/100 and 1/3–1 saturated DWSF concentration, respectively.

IBR system construction and analysis

In this study, three parameters of photosynthetic rate, photosynthetic fluorescence, photosynthetic pigment and oxidative stress were selected to construct IBR system to comprehensively evaluate the dose-effect relationship of DWSF on the physiological characteristics of T. hemprichii. Photosynthetic rate and photosynthetic fluorescence IBR system Seven photosynthetic physiological parameters, RR, NPR, F v / F m, NPQ, ETR, q P and Φ PSII q, were selected as biomarkers. Chla, Chlb, Chl (a + b), Chla/Chlb, C x and C T were selected as biomarkers in the IBR system of photosynthetic pigments. In the oxidative stress IBR system, six antioxidant enzyme activities (POD, SOD, CAT, APX, H 2 O 2 and MDA) and related parameters of oxidative stress products were selected as biomarkers.

The analysis results of physiological changes of T. hemprichii leaves under different concentrations of DWSF exposure stress based on different IBR systems are shown in Figure 5 and Figure 6. It can be seen from Figure 5 that with the increase of DWSF concentration, the radiation length corresponding to each biomarker and the area of the final enclosed star map showed an increasing trend. The IBR value of each treatment in Figure 6 is the specific value of the area of the polygon surrounded by the radiation lines of each biomarker in the corresponding IBR system in Figure 5. The greater the IBR value, the greater the impact on the grass. Under the IBR system of photosynthetic rate and photosynthetic fluorescence, the IBR values of T. hemprichii treated with different concentrations of DWSF increased step by step: the IBR value of CK treatment was the lowest, which was 0; when the DWSF concentration is in the range of 1/100 - 1/10 of the saturated concentration, the IBR value changes in the range of 2 - 5. When DWSF concentration ≥ 1/3 saturated concentration, the IBR value is maintained at a higher value of about 13. In the IBR system of photosynthetic pigments, with the increase of DWSF concentration, the IBR value of T. hemprichii decreased first and then increased. The inflection point appeared at the DWSF concentration of 1/100 saturated concentration, and when the DWSF concentration was ≤ 1/30 saturated concentration, the IBR value was lower (<1). Under the oxidative stress IBR system, the IBR value of T. hemprichii gradually increased with the increase of DWSF concentration.

The results of correlation analysis between IBR value and DWSF concentration in seawater showed that there was a positive correlation between them under each IBR system (Figure 7), that is, the IBR value increased with the increase of DWSF concentration, and the positive correlation between photosynthetic pigment and oxidative stress IBR system reached a very significant level (P < 0.01).

4. Discussion

Effects of DWSF on growth and photosynthetic physiology of Thalassia hemprichii

Aromatic hydrocarbon components and their derivatives (such as anthracene, benzene, naphthalene and phenanthrene) that are more soluble in water in petroleum and its products tend to be enriched in the hydrophobic thylakoid membrane of plants after being absorbed by marine plants, which will break the balance between cell membrane ions and affect the transfer of electrons in plant photosynthesis (Wang et al., 2004; Huang et al., 2010). At the same time, the reactive oxygen species produced by plant antioxidant stress will accumulate in large quantities, destroy its cell membrane system, cause damage to chloroplasts and other organelles, and then affect its photosynthetic pigment content and growth rate (Thorhaug and Marcus, 1987). Bate and Crafford (1985) found that the soluble components of lubricating oil had a significant inhibitory effect on the photosynthesis of Phaeodactylum tricornutum, Tetraselmis suecica, Dunaliella salina and Anacystis marina. Stepaniyan (2008) conducted a short-term toxicity test on Laminaria japonica, (Fucus vesiculosus and Porphyra haitanensis with crude oil and found that the photosynthesis of several algae was inhibited. This study found that with the increase of DWSF concentration in seawater, leaf growth was inhibited only at relatively high concentrations, Chlb content generally decreased, and most photosynthetic fluorescence parameters (F m, F v / F m, ETR, q P and Φ PSII) showed a declining tendency. By contrast, Chla and total pigment concentrations presented a biphasic pattern of initial stimulation followed by inhibition, while carotenoid levels remained stable across treatments. Combined with the increase of antioxidant enzyme activity and oxidative stress product content, it was shown that DWSF, like crude oil or other petroleum products, can also cause oxidative stress and inhibit photosynthetic efficiency and photosynthetic capacity of seagrass (Li et al., 2021).

Some studies have found that the effect of petroleum hydrocarbons in seawater on marine plants will appear the phenomenon of low-dose stimulation and high-dose inhibition, that is, low concentration of petroleum hydrocarbons will produce the phenomenon of low-dose stimulation and high-dose inhibition on plants, and only when the concentration is higher than a certain threshold, it will inhibit its growth (El-Sheekh et al., 2000; Shen et al., 2006, 2023). For example, Li et al. (2016) showed that low concentration of DWSF could promote the growth of Chlorella vulgaris, while high concentration would inhibit its growth, and the inflection point appeared at 0.90 mg/L DWSF. Wang et al. (2011) found that the photosynthetic rate of Ulva pertusa showed a trend of low concentration promotion and high concentration inhibition in response to different concentrations of three petroleum hydrocarbons, and the inflection point appeared in the range of 15–25 mg/L. The results of this study showed that with the increase of DWSF concentration, the physiological parameters such as NPR and Chla content of T. hemprichii also showed a similar trend of the phenomenon of low-dose stimulation and high-dose inhibition, and the inflection point appeared at 1/100-1/30 saturated DWSF concentration, that is, the DWSF content was 0.84-2.80 mg/L range. It shows that the sensitivity of T. hemprichii to DWSF is comparable to that of phytoplankton, but its tolerance to petroleum hydrocarbon pollution is lower than that of large marine algae. The reason for this phenomenon may be that the relevant physiological indexes of T. hemprichii have certain resistance to petroleum hydrocarbon pollution. When the concentration of DWSF is low, the stress effect is limited, but it is promoted by providing sufficient carbon and nitrogen sources.

Changes in photosynthetic fluorescence parameters of plants can be used to evaluate the stress effects on their photosynthetic physiology (Ryzhik and Makarov, 2019). In this study, with the increase of DWSF concentration, the photosynthetic fluorescence parameters such as F v / F m, ETR, q P and Φ PSII in the leaves of T. hemprichii showed different degrees of reduction, indicating that it suffered a certain degree of stress pressure (Wang et al., 2022). The F v / F m of plant leaves is often used to measure the level of chloroplast PSII activity under various environmental stresses (Wuthirak et al., 2016). Under normal environmental conditions, F v / F m is generally maintained at about 0.76 (Hader et al., 1996), while under stress conditions, the parameter will be significantly reduced (Zhang and Huang, 2009; Lu et al., 2022), and Fv/Fm< 0.44 indicates that the PSII reaction center is completely inactivated (Schansker and van Rensen, 1999). In this study, when the seawater DWSF concentration ≤ 1/10 saturated concentration, the F v / F m of seagrass leaves did not decrease significantly compared with CK (P >0.05); when DWSF concentration reached saturation (i.e. BH treatment), the F v / F m of leaves was still as high as 0.68, which was only 15% lower than that of CK, indicating that T. hemprichii had strong tolerance to DWSF. At the same time, the concentration of DWSF in BH/100 treatment in this study has reached 0.84 mg/L, which is 68% higher than the maximum value (0.50 mg/L) of the fourth type of seawater petroleum content required by the Seawater Quality Standard (GB 3097-1997). At this time, most physiological indexes of T. hemprichii have no significant difference compared with CK (P >0.05), which again shows that T. hemprichii has strong resistance to petroleum hydrocarbon pollution.

Effects of DWSF on antioxidant enzyme activity and oxidative stress products of Thalassia hemprichii

Plant antioxidant system has dual functions of responding to stress and regulating reactive oxygen signal transduction (Lu et al., 2022). The by-product reactive oxygen species (ROS, including superoxide anion, H 2 O 2, singlet oxygen and hydroxyl radicals) produced by aerobic metabolism of green plants can cause oxidative damage to plants. Plants have also evolved a fine antioxidant mechanism to scavenge reactive oxygen species (Li et al., 2021; Lu et al., 2022). Under normal circumstances, the antioxidant system in plants can remove excessive ROS in a timely manner; under stress, the generation rate of ROS in plants is accelerated, the scavenging ability of antioxidant system is relatively insufficient, and the dynamic balance of ROS is broken, resulting in accumulation (Yang et al., 2016). Accumulated ROS not only cause oxidative stress and oxidative damage but also participate in a variety of information transmission processes and activate plant stress response mechanisms (Li et al., 2018). Factors such as the strength and duration of stress and their own tolerance will affect the change trend of antioxidant enzyme activity and oxidative stress product content in plants (Yang et al., 2016; Li et al., 2018, 2021; Lu et al., 2022). Li et al. (2021) found that under the stress of 5 mg/L petroleum hydrocarbon aqueous solution, the SOD activity of T. hemprichii increased first and then decreased, while the POD activity gradually decreased with the prolongation of stress time. The results of this study showed that with the increase of DWSF concentration, the contents of two oxidative stress products (H 2 O 2 and MDA) and the activities of four antioxidant enzymes (POD, SOD, CAT and APX) in the leaves of T. hemprichii increased. The increase in the content of oxidative stress products indicates that the pollution stress of DWSF leads to the accumulation of ROS in T. hemprichii and the peroxidation damage of membrane lipids, because H 2 O 2 is the intermediate product of reactive oxygen species when the electron transfer in the plant is inhibited under stress (González et al., 2009). MDA is the product of lipid peroxidation in plant body caused by ROS (Li et al., 2018). The increase of antioxidant enzyme activity indicates that the accumulation of ROS in T. hemprichii activates its stress response mechanism, which up-regulates the expression of antioxidant enzyme genes to remove the extra generated ROS (Liu et al., 2011). At the same time, due to the strong tolerance of T. hemprichii to DWSF, the stress effect did not exceed the anti-stress regulation ability of seagrass during the concentration and culture period set in this study. Therefore, there was no significant decrease in antioxidant enzyme activity caused by the decrease of plant metabolic level (Aguirre-Macedo et al., 2015). The results of this study also showed that there were significant differences in the stress-induced sensitivity of different types of antioxidant enzymes in the leaves of T. hemprichii. CAT and APX were more sensitive, followed by SOD, and POD was less sensitive.

The dose-effect IBR evaluation system of DWSF on the physiological effects of Thalassia hemprichii

The IBR index is widely used to assess organismal health and integrated biological stress responses under contaminant exposure (Niyogi et al., 2001; Chen et al., 2022; Xie et al., 2024). Radar-star diagrams of standardized biomarker scores enable intuitive visualization of dose-dependent physiological shifts in T. hemprichii following DWSF exposure.

Existing petroleum-toxicity IBR investigations on marine organisms are largely based on antioxidant biomarkers of invertebrate species (Jiang et al., 2015; Chen et al., 2022). By comparison, seagrass-relevant IBR studies remain scarce and are mostly conducted on temperate species such as Zostera marina subjected to thermal, salinity or heavy-metal stress, usually employing a single combined biomarker panel. Temperate seagrasses commonly exhibit relatively monotonic stress responses, whereas tropical T. hemprichii may show biphasic low-dose stimulation-high-dose inhibition patterns under petroleum pollution.

Three independent IBR systems were constructed in the present work, corresponding to photosynthetic rate-chlorophyll fluorescence, photosynthetic pigments and oxidative-stress pathways. Each system reflects a distinct physiological dimension: light utilization and electron-transport performance, light-harvesting pigment composition, and antioxidant-mediated membrane protection. The photosynthetic-pigment and oxidative-stress IBR values were strongly and positively correlated with DWSF concentration. Pigment measurement is low-cost and operationally convenient for large-scale monitoring, while the oxidative-stress system provides mechanistic evidence of pollutant-induced oxidative injury. Despite an overall upward trend with rising DWSF levels, the photosynthesis-fluorescence IBR was affected by biphasic or plateau-type biomarker responses at high concentrations, which weakened its linear correlation. The pigment-based and oxidative-stress IBR systems are therefore preferred for DWSF toxicity assessment, and the photosynthetic-fluorescence module can serve as a supplementary indicator.

One caveat of this study is that toxicity was assessed based on bulk TPH concentration rather than speciated hydrocarbon data. Since DWSF is a complex mixture, different alkane and aromatic components may exert distinct toxic effects on seagrass physiology. Future experiments combining chemical component analysis are warranted to further unpack the compound-specific toxic mechanisms. Another experimental limitation of this semi-static exposure assay is the absence of periodic water-phase DWSF concentration measurements during incubation. Pollutant losses via volatilization, adsorption and microbial degradation could reduce the actual exposure concentration within each 48-hour renewal cycle. Consequently, the effective time-weighted exposure level may be slightly lower than the nominal concentration, which could potentially lead to mild underestimation of the toxicity threshold of DWSF for T. hemprichii.

It should also be noted that carotenoid (Cx) concentrations did not differ significantly across DWSF treatments. Cx was retained in the pigment-based IBR system owing to its fundamental photoprotective and antioxidant roles, to maintain the physiological integrity of the pigment biomarker panel, even though this relatively stable biomarker may slightly reduce overall index sensitivity.

5. Conclusions

With the increase of DWSF concentration in seawater, the growth rate, dark respiration rate (RR), chlorophyll b (Chlb) content, maximum fluorescence intensity (F m), maximum photosynthetic efficiency (F v / F m), apparent photosynthetic electron transport rate (ETR), photochemical quenching coefficient (qP) and actual photochemical efficiency (Φ PSII) of T. hemprichii leaves showed a decreasing trend, while the net photosynthetic rate (NPR), chlorophyll a (Chla) content, total photosynthetic pigment (CT) content and initial fluorescence intensity (F 0) showed the phenomenon of low-dose stimulation and high-dose inhibition. The non-photochemical quenching coefficient (NPQ), antioxidant enzyme activity and oxidative stress product content showed an increasing trend, reflecting that with the increase of DWSF concentration, the pollution stress of T. hemprichii was aggravated.

The inflection point of important physiological parameters such as NPR and Chla content in the leaves of T. hemprichii the phenomenon of low-dose stimulation and high-dose inhibition appeared at 1/100 - 1/30 saturated DWSF concentration. When the DWSF concentration reached saturation, the F v / F m of the leaves was still as high as 0.68 and the stress effect did not exceed the stress regulation ability of T. hemprichii, indicating that T. hemprichii had strong tolerance to DWSF, and the stress effect was limited when the DWSF concentration was low.

The IBR system based on photosynthetic rate and three biomarkers of photosynthetic fluorescence, photosynthetic pigment and oxidative stress can comprehensively evaluate the dose-effect relationship of DWSF on the physiological characteristics of T. hemprichii. The IBR value of photosynthetic pigment and oxidative stress system has a very significant positive correlation with the content of DWSF in seawater (P < 0.01), which is more suitable for the comprehensive evaluation of the stress effect of petroleum hydrocarbon pollution on T. hemprichii.

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor declared a past co-authorship with the authors ZN and YS.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by Natural Science Foundation of China (No.42466007), Joint Fund Project of National Natural Science Foundation of China (No.U24A20637), Scientific Research Foundation of Hainan Tropical Ocean University (No. RHDRC202308).

Data availability

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Author contributions

SM: Methodology, Conceptualization, Writing – original draft, Writing – review & editing. MZ: Methodology, Writing – original draft, Writing – review & editing, Conceptualization. KW: Data curation, Methodology, Writing – original draft. ZN: Funding acquisition, Writing – review & editing. YS: Funding acquisition, Writing – review & editing, Conceptualization. YL: Writing – original draft, Data curation. YF: Investigation, Writing – original draft. QH: Writing – review & editing, Methodology.

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Citation

Ma, S., et al. (2026). Effect of diesel oil water-soluble fraction on physiological characteristics of Thalassia hemprichii and its integrated biomarker response. Frontiers in Ecology and Evolution, Volume 14. https://doi.org/
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Copyright © 2026 Ma, Zhao, Wang, Ni, Shi, Li, Feng and Han.

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*Correspondence: Zhen Ni; Yunfeng Shi

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