Antioxidant Response and Oxidative Stress in the Respiratory Tree of Sea Cucumber ( Apostichopus japonicus ) Following Exposure to Crude Oil and Chemical Dispersant

: Sea cucumber ( Apostichopus japonicus ) is mainly cultured in the coastal zone, where it is easily threatened by accidental oil spills. Chemical dispersant is one of the e ﬃ cient oil spill responses for mitigating the overall environmental damage of oil spills. However, the impact of crude oil and chemical dispersants on sea cucumber is less well known. Hence, the present study focused on exploring the antioxidant response and oxidative stress in the respiratory tree of sea cucumber following exposure to GM-2 chemical dispersant (DISP), water-accommodated fractions (WAF), and chemically enhanced WAF (CEWAF) of Oman crude oil for 24 h. Results manifested that WAF exposure caused a signiﬁcant increase in the reactive oxygen species (ROS) level (5.29 ± 0.30 AU · mgprot − 1 ), and the e ﬀ ect was much more obvious in CEWAF treatment (5.73 ± 0.16 AU · mgprot − 1 ). Total antioxidant capacity (T-AOC), as an important biomarker of the antioxidant defense capacity, showed an increasing trend following WAF exposure (0.95 ± 0.12 U · mgprot − 1 ) while a signiﬁcant reduction in T-AOC was observed following CEWAF exposure (0.23 ± 0.13 U · mgprot − 1 ). Moreover, we also evaluated the oxidative damage of the macromolecules (DNA, protein, and lipid), and our results revealed that the presence of chemical dispersant enhanced oxidative damage caused by crude oil to sea cucumber.


Introduction
The growing global production and consumption of crude oil and its refined products have generated public concern about the effects of potential oil spills on marine ecosystems. Oil slicks from an accidental oil spill can reach coastal habitats through the action of wind, waves, and currents [1][2][3], and subsequently cause disastrous impacts on coastal zone and human activities. For instance, the 2010 Xingang Port oil spill in Dalian (China) caused more than 1500 t crude oil spilled into the Yellow Sea, resulting in severe losses of marine aquaculture in Liaoning Province. The 2011 Bohai bay oil spill, one of the worst oil pollution incidents along the Chinese coastline, led to more than 840 km 2 of polluted area and direct economic losses up to CNY12.56 billion in Hebei, Shandong, and Liaoning Province [4]. Chemical dispersants can break up oil slicks into tiny droplets (≤70 µm) in the water column, and then enhance the natural processes of oil slicks, resulting in reducing the amount of oil slicks that might reach coastal habitats [5,6]. Therefore, chemical dispersants have been regarded as one of the efficient Park, Dalian, China, and pre-filtered through sterile 0.45 µm filters. WAF, CEWAF (Oman crude oil + GM-2), and DISP (GM-2 alone) solutions were prepared according to the Chemical Response to Oil Spills: Ecological Effects Research Forum (CROSERF) method with some modifications [8,28]. Briefly, WAF solution was prepared with natural seawater at an oil loading rate of 5 g·L −1 in a 10 L glass aspirator bottle (leaving approx. 20% headspace) sealing instantly with a rubber stopper after loading Oman crude oil. The oil-seawater mixture was mixed with a Teflon-coated stirring bar for 18 h and settled for 6 h in the dark at 16.0 ± 0.5 • C to separate the WAF solution from the oil phase. CEWAF solution preparation was performed with natural seawater at an oil loading rate of 5 gL −1 in a 10 L glass aspirator bottle (leaving approx. 20% headspace). The oil-seawater mixture was mixed with a Teflon-coated stirring bar to form a vortex steadily and then, GM-2 chemical dispersant was sequentially delivered into the center of the vortex at a dispersant-to-oil ratio (DOR) of 1:5 (m/m). The mixture was stirred for 18 h and settled for 6 h in the dark at 16.0 ± 0.5 • C to separate the CEWAF solution from the oil phase. DISP solution preparation followed the same steps as the CEWAF preparation without adding Oman crude oil. All the solutions were freshly prepared prior to the exposure experiments.

Sea Cucumber Maintenance and Acute Toxicity Tests
Sea cucumber (Apostichopus japonicus, Selenka), with an average wet weight of 56.39 ± 9.38 g, were sourced from Pikou sea cucumber aquaculture zoning, Dalian, China, and acclimatized in an indoor recirculating aquaculture system with a density of 15 per tank (around 60 L capacity). The maintenance conditions were listed as follows: temperature 16.0 ± 0.5 • C, pH 7.9 ± 0.2, salinity 32.0 ± 1.0 psu, dissolved oxygen 7.1 ± 0.3 mg·L −1 , and a photoperiod of 14 h light/10 h dark with continuous aeration. Sea cucumber were fed once with formulated feeds per day, and the residual food and feces were siphoned. After a 7-d acclimation period, sea cucumber were randomly allocated to four treatment groups: three exposure groups (DISP, WAF, and CEWAF) and a Control group (pre-filtered natural seawater only) (Supplementary Materials Figure S1). After a 24-h exposure period, the respiratory trees of sea cucumber were dissected and washed with pre-cold 100 mM potassium phosphate (PBS) buffer (pH 7.4). Then, the samples were immediately frozen using liquid nitrogen in the preservation tubes containing 100 mM PBS buffer at a ratio of 1:9 (m/v) for subsequent analysis of biochemical indicators.

Reactive Oxygen Species (ROS) Production
The ROS production was determined using a 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) method [29] with the Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocol. Briefly, fresh tissues were washed with pre-cold 100 mM PBS buffer (pH 7.4) twice and then homogenized in pre-cold PBS buffer. The homogenate was centrifuged for 20 min at 500× g, and the precipitate was collected. Then, the precipitate was incubated with 100 µL PBS (pH 7.4) and 50 µL DCFH-DA (1 mM) over 60 min at 37 • C. The fluorescence intensity was monitored with excitation at 485 nm and emission at 525 nm using a SpectraMax M5 multimode microplate reader (Molecular Devices, USA). The ROS level was normalized to protein concentration for each sample, which was measured at 595 nm with the Total Protein Assay Kit (Bradford method) (Nanjing Jiancheng Bioengineering Institute, China). The ROS level was expressed in arbitrary units per mg protein (AU·mgprot −1 ).

Antioxidant Capacity
The total antioxidant capacity (T-AOC) was measured using the ferric reducing ability of plasma (FRAP) method [30] with the Total Antioxidant Capacity Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). In brief, tissue sample with PBS buffer was homogenized on the ice and centrifuged for 15 min at 3000 rpm, and then the supernate was collected. 10 µL of supernate, 20 µL of peroxidase application solution, and 170 µL of ABTS working solution were added into a 96 well microplate and mixed fully. The mixture was incubated for 6 min at room temperature (24 • C). The optical density (OD) value was monitored at 520 nm using a microplate ultraviolet-visible (UV-Vis) spectrophotometer (BioTek, USA). The T-AOC was expressed as units per mg protein (U·mgprot −1 ).

Oxidative Damage Assessment
Oxidative DNA damage was assessed according to an 8-hydroxy-2 -deoxyguanosine (8-OHdG) method [31] with the 8-OHdG enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bioengineering Institute, China). According to the manufacturer's instruction, 50 µL of the tissue sample homogenate was added with 50 µL of the horseradish peroxidase (HRP) conjugated 8-OHdG antibody into a microtiter ELISA plate well, which had been pre-coated with an antibody specific for 8-OHdG. The plate was incubated for 1 h at 37 • C. After incubation, the well was washed five times with 300 µL washing buffer. Then, chromogenic solutions were added and incubated at 37 • C for 15 min. The enzymatic color reaction was terminated by adding 50 µL of 2 M H 2 SO 4 . The OD value of each well was detected at 450 nm. The 8-OHdG level was determined for each sample from a standard curve and expressed as ng per mg protein (ng·mgprot −1 ).
Protein oxidation was determined based on the level of protein carbonyls (PCO) according to the 2,4-dinitrophenylhydrazide (DNPH) method [32,33] with the Protein Carbonyl Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). Briefly, 100 µL of the tissue sample homogenate was incubated with 10 mM DNPH (in 2 N HCl) in the dark at 37 • C for 30 min. An equivalent volume of trichloroacetic acid (TCA) was added and centrifuged for 10 min at 12,000 rpm. The precipitate was washed four times with ethanol/ ethyl acetate (1:1, v/v) mixture. The final precipitate was redissolved in 6 M guanidine hydrochloride and incubated at 37 • C for 15 min. After a 15-min centrifugation at 12,000 rpm, the OD value of the final supernate was measured at 370 nm. The PCO level was calculated based on the protein concentration for each sample and expressed as nanomoles per mg protein (nmol·mgprot −1 ).
Lipid peroxidation was evaluated using a malondialdehyde (MDA) method described by Buege et al. [34] with the Malondialdehyde Assay Kit (thiobarbituric acid (TBA) method)) following the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, China). Briefly, 500 µL of the tissue sample homogenate and 500 µL of TBA were added into a microcentrifuge tube and mixed fully. The reaction mixture was incubated at 95 • C for 60 min and cooled to room temperature in an ice bath for 10 min. The OD value of the reaction mixture was measured at 532 nm. The MDA concentration was expressed as nanomoles per mg protein (nmol·mgprot −1 ).

Integrated Biomarker Response (IBR) Index
To further compare the relative values of oxidative stress responses in DISP, WAF, and CEWAF treatments, integrated biomarker response (IBR) index was applied as described by Beliaeff Benoit et al. [35] and modified by Sanchez Wilfried et al. [36] and Vieira et al. [37]. In the present study, the deviation between biomarkers measured in different treatments (X i ) were compared to those measured in the Control (X 0 ). For each biomarker, the ratio of X i and X 0 was log-transformed (Y i ). Then, the general mean (µ) and standard deviation (s) were calculated in each group. Y i values were standardized by the formula: Z i = (Y i − µ)/s and the difference between Z i and Z 0 (the Control) was used to define the biomarker deviation index (A). To obtain an IBR index, the A value of each biomarker was calculated for each treatment and the IBR index was determined by the sum of the absolute values of A.

Chemicals Analysis
Total petroleum hydrocarbons (TPH) concentration was measured by ultraviolet spectrometry using the Standardization Administration of China (SAC) GB 17378.4-2007 method with some modifications as described in our previous work [38,39]. Briefly, 200 mL water sample was extracted with 20 mL n-hexane (with a transmittance over 90% at 225 nm wavelength) in a separatory funnel. The absorbance of n-hexane extract was detected at 225 nm by the UV-Vis spectrophotometer in a quartz cuvette. The TPH concentration was determined based on the calibration curve (WAF: r 2 = 0.991; CEWAF: r 2 = 0.999; Figure 1A). Moreover, 16 priority PAHs listed by US Environmental Protection Agency (EPA) were also analyzed in the present study as described in our previous work [38], including naphthalene (Nap), acenaphthylene (Acy), acenaphthene (Ace), fluorene (Fle), phenanthrene (Phe), anthracene (Ant), fluoranthene (Fla), pyrene (Pyr), benzo [ [40]. Water sample was pre-processed according to the EPA 3510C liquid-liquid extraction method [41]. Cleanup of the concentrate was performed based on the EPA 3630C silica gel cleanup method [42]. Then, PAHs were analyzed using an Agilent 7890B gas chromatography coupled 5977A mass selective detector according to ISO 28540:2011 [43].

Analytical Chemistry
The concentrations of TPH were 4.25 ± 0.07 and 57.81 ± 1.15 mg·L −1 in WAF and CEWAF solutions, respectively, indicating that the TPH level in CEWAF solution was 13.60 fold higher than that in WAF solution. The concentrations of total 16 priority PAHs (ΣPAHs) in WAF and CEWAF solutions were 2.25 ± 0.43 and 5.68 ± 0.82 μg·L -1 , respectively. The ΣPAHs concentration in CEWAF solution was 2.52 fold higher than that in WAF solution. Moreover, the proportions (%) of each PAH showed various changes between WAF and CEWAF solutions. In WAF solution, the most abundant components were lower molecular weight (LMW) PAHs (2-ringed) accounting for 57.9% of ΣPAHs, followed by higher molecular weight (HMW) PAHs, including 3-ringed (15.5%), 4-ringed (13.6%) and ≥5-ringed (13.0%) PAHs. In CEWAF solution, the LMW PAHs (46.3%) were still the most abundant components but showed a relatively lower proportion than that in WAF solution, accompanied by a corresponding increase in the proportion of HMW PAHs, including 3-ringed PAHs accounting for 18.6%, 4-ringed PAHs accounting for 17.7%, and ≥5-ringed PAHs accounting for 17.4% ( Figure 1B).

Statistical Analysis
All data were presented as the mean ± standard deviation (SD). Statistical difference between the treatments and the Control was analyzed via the one-way analysis of variance (one-way ANOVA). Significant differences were accepted when p < 0.05. The asterisks (*, **, or ***) in the graphics denoted statistically significant difference from the Controls (p < 0.05, 0.01, or 0.001, respectively). Statistical analysis and graph plotting were performed using SigmaPlot Ver 14.0 (Systat Software, Inc., USA).

Analytical Chemistry
The concentrations of TPH were 4.25 ± 0.07 and 57.81 ± 1.15 mg·L −1 in WAF and CEWAF solutions, respectively, indicating that the TPH level in CEWAF solution was 13.60 fold higher than that in WAF solution. The concentrations of total 16 priority PAHs (ΣPAHs) in WAF and CEWAF solutions were 2.25 ± 0.43 and 5.68 ± 0.82 µg·L −1 , respectively. The ΣPAHs concentration in CEWAF solution was 2.52 fold higher than that in WAF solution. Moreover, the proportions (%) of each PAH showed various changes between WAF and CEWAF solutions. In WAF solution, the most abundant components were lower molecular weight (LMW) PAHs (2-ringed) accounting for 57.9% of ΣPAHs, followed by higher molecular weight (HMW) PAHs, including 3-ringed (15.5%), 4-ringed (13.6%) and ≥5-ringed (13.0%) PAHs. In CEWAF solution, the LMW PAHs (46.3%) were still the most abundant components but showed a relatively lower proportion than that in WAF solution, accompanied by a corresponding increase in the proportion of HMW PAHs, including 3-ringed PAHs accounting for 18.6%, 4-ringed PAHs accounting for 17.7%, and ≥5-ringed PAHs accounting for 17.4% ( Figure 1B).

Survival Rates
During the 24-h acute exposure period, WAF, CEWAF or DISP treatments did not cause a reduction in the survival rates of sea cucumber compared to the Control (Chi-square, p > 0.05, Supplementary Materials Figure S2).

ROS Levels
No statistical difference was observed between the DISP treatment (4.53 ± 0.75 AU·mgprot −1 ) and the Control (4.65 ± 0.21 AU·mgprot −1 ) (one-way ANOVA, p = 0.94 > 0.05), manifesting that acute exposure to DISP had no obvious impact on the ROS level in sea cucumber ( Figure 2). The ROS levels in WAF and CEWAF treatments were 5.29 ± 0.30 and 5.73 ± 0.16 AU·mgprot −1 , respectively, indicating that both WAF and CEWAF exposure caused a significant increase in ROS levels compared with the Control (one-way ANOVA, p = 0.03 < 0.05 and p < 0.001 for WAF and CEWAF, respectively). Moreover, the ROS level in CEWAF treatment was also much higher than that in the WAF (one-way ANOVA, p = 0.002 < 0.01).

Survival Rates
During the 24-h acute exposure period, WAF, CEWAF or DISP treatments did not cause a reduction in the survival rates of sea cucumber compared to the Control (Chi-square, p > 0.05, Appendix A Figure S2).

ROS Levels
No statistical difference was observed between the DISP treatment (4.53 ± 0.75 AU·mgprot -1 ) and the Control (4.65 ± 0.21 AU·mgprot -1 ) (one-way ANOVA, p = 0.94 > 0.05), manifesting that acute exposure to DISP had no obvious impact on the ROS level in sea cucumber ( Figure 2). The ROS levels in WAF and CEWAF treatments were 5.29 ± 0.30 and 5.73 ± 0.16 AU·mgprot -1 , respectively, indicating that both WAF and CEWAF exposure caused a significant increase in ROS levels compared with the Control (one-way ANOVA, p = 0.03 < 0.05 and p < 0.001 for WAF and CEWAF, respectively). Moreover, the ROS level in CEWAF treatment was also much higher than that in the WAF (one-way ANOVA, p = 0.002 < 0.01).

Figure 2.
Reactive oxygen species (ROS) levels in the respiratory tree of sea cucumber following GM-2 chemical conventional dispersant (DISP, gray filled), water-accommodated fractions (WAF, dark-gray filled), and chemically enhanced WAF (CEWAF, light-gray filled) of Oman crude oil. The Control was the pre-filtered natural seawater only (black filled). Asterisks denote significant differences between the treatments and the Control. Dark traits denote significant differences among the treatments (one-way analysis of variance (ANOVA)).

Antioxidant Capacity
To assess the antioxidant defense capacity in the respiratory tree of sea cucumber following the DISP, WAF, and CEWAF exposure, T-AOC was measured in the present study ( Figure 3). Results showed that compared with the Control (0.82 ± 0.14 U·mgprot −1 ), DISP had no significant impact on the T-AOC (0.79 ± 0.13 U·mgprot −1 ; one-way ANOVA, p = 0.99 > 0.05). WAF exposure caused a slight increase in the T-AOC (0.95 ± 0.12 U·mgprot -1 ) but without significant difference compared with the Control (one-way ANOVA, p = 0.65 > 0.05); while CEWAF exposure induced an extremely significant decrease in the T-AOC (0.23 ± 0.13 U·mgprot −1 ) (one-way ANOVA, p < 0.001). Meanwhile, the T-AOC in CEWAF treatment was also much lower than that in WAF treatment (one-way ANOVA, p < 0.001).

Figure 2.
Reactive oxygen species (ROS) levels in the respiratory tree of sea cucumber following GM-2 chemical conventional dispersant (DISP, gray filled), water-accommodated fractions (WAF, dark-gray filled), and chemically enhanced WAF (CEWAF, light-gray filled) of Oman crude oil. The Control was the pre-filtered natural seawater only (black filled). Asterisks (*, or ***) denote significant differences between the treatments and the Control (p < 0.05 or 0.001, respectively). Dark traits denote significant differences among the treatments (one-way analysis of variance (ANOVA)).

Antioxidant Capacity
To assess the antioxidant defense capacity in the respiratory tree of sea cucumber following the DISP, WAF, and CEWAF exposure, T-AOC was measured in the present study ( Figure 3). Results showed that compared with the Control (0.82 ± 0.14 U·mgprot −1 ), DISP had no significant impact on the T-AOC (0.79 ± 0.13 U·mgprot −1 ; one-way ANOVA, p = 0.99 > 0.05). WAF exposure caused a slight increase in the T-AOC (0.95 ± 0.12 U·mgprot −1 ) but without significant difference compared with the Control (one-way ANOVA, p = 0.65 > 0.05); while CEWAF exposure induced an extremely significant decrease in the T-AOC (0.23 ± 0.13 U·mgprot −1 ) (one-way ANOVA, p < 0.001). Meanwhile, the T-AOC in CEWAF treatment was also much lower than that in WAF treatment (one-way ANOVA, p < 0.001).  Control was the pre-filtered natural seawater only (black filled). Asterisks denote significant differences between the treatments and the Control. Dark traits denote significant differences among the treatments (one-way ANOVA).

Oxidative Damage
To further evaluate the oxidative damage of the macromolecules (DNA, proteins, and lipids) after DISP, WAF, and CEWAF exposure, the levels of 8-OHdG, PCO, and MDA in the respiratory tree of sea cucumber were detected in the present study. For oxidative DNA damage, the standard curve of 8-OHdG level was fitted using the nonlinear logistic model with four parameters (r 2 = 0.972; Figure 4A). Results showed that no significant differences were found for 8-OHdG levels for the three treatments compared with the Control (one-way ANOVA, p = 0.68, 0.61, and 0.18 > 0.05 for DISP, WAF, and CEWAF, respectively; Figure 4B), indicating that none of DISP, WAF, or CEWAF exposure caused significant change in the levels of oxidative DNA damage. Regarding the protein oxidation, the levels of PCO were determined ( Figure 4C). Results manifested that both DISP and WAF exposure induced a slight increase in the PCO levels but without significant differences compared with the Control (one-way ANOVA, p = 0.92 and 0.19 > 0.05 for DISP and WAF, respectively); while CEWAF exposure produced a significant elevation of the PCO level (one-way ANOVA, p = 0.04 < 0.05). As for lipid peroxidation, MDA contents were measured ( Figure 4D). Results revealed that the MDA contents in WAF and CEWAF treatments were much higher than that in the Control (one-way ANOVA, p < 0.01); while the MDA content in DISP treatment was subtly lower than that in the Control but without significant difference (one-way ANOVA, p = 0.67 > 0.05). Moreover, the MDA content in CEWAF treatment was also much higher than that in WAF treatment (one-way ANOVA, p < 0.001). , water-accommodated fractions (WAF, dark-gray filled) and chemically enhanced WAF (CEWAF, light-gray filled) of Oman crude oil. The Control was the pre-filtered natural seawater only (black filled). Asterisks (***) denote significant differences between the treatments and the Control (p < 0.001). Dark traits denote significant differences among the treatments (one-way ANOVA).

Oxidative Damage
To further evaluate the oxidative damage of the macromolecules (DNA, proteins, and lipids) after DISP, WAF, and CEWAF exposure, the levels of 8-OHdG, PCO, and MDA in the respiratory tree of sea cucumber were detected in the present study. For oxidative DNA damage, the standard curve of 8-OHdG level was fitted using the nonlinear logistic model with four parameters (r 2 = 0.972; Figure 4A). Results showed that no significant differences were found for 8-OHdG levels for the three treatments compared with the Control (one-way ANOVA, p = 0.68, 0.61, and 0.18 > 0.05 for DISP, WAF, and CEWAF, respectively; Figure 4B), indicating that none of DISP, WAF, or CEWAF exposure caused significant change in the levels of oxidative DNA damage. Regarding the protein oxidation, the levels of PCO were determined ( Figure 4C). Results manifested that both DISP and WAF exposure induced a slight increase in the PCO levels but without significant differences compared with the Control (one-way ANOVA, p = 0.92 and 0.19 > 0.05 for DISP and WAF, respectively); while CEWAF exposure produced a significant elevation of the PCO level (one-way ANOVA, p = 0.04 < 0.05). As for lipid peroxidation, MDA contents were measured ( Figure 4D). Results revealed that the MDA contents in WAF and CEWAF treatments were much higher than that in the Control (one-way ANOVA, p < 0.01); while the MDA content in DISP treatment was subtly lower than that in the Control but without significant difference (one-way ANOVA, p = 0.67 > 0.05). Moreover, the MDA content in CEWAF treatment was also much higher than that in WAF treatment (one-way ANOVA, p < 0.001).  Control was the pre-filtered natural seawater only (black filled). Asterisks denote significant differences between the treatments and the Control. Dark traits denote significant differences among the treatments (one-way ANOVA).

Oxidative Damage
To further evaluate the oxidative damage of the macromolecules (DNA, proteins, and lipids) after DISP, WAF, and CEWAF exposure, the levels of 8-OHdG, PCO, and MDA in the respiratory tree of sea cucumber were detected in the present study. For oxidative DNA damage, the standard curve of 8-OHdG level was fitted using the nonlinear logistic model with four parameters (r 2 = 0.972; Figure 4A). Results showed that no significant differences were found for 8-OHdG levels for the three treatments compared with the Control (one-way ANOVA, p = 0.68, 0.61, and 0.18 > 0.05 for DISP, WAF, and CEWAF, respectively; Figure 4B), indicating that none of DISP, WAF, or CEWAF exposure caused significant change in the levels of oxidative DNA damage. Regarding the protein oxidation, the levels of PCO were determined ( Figure 4C). Results manifested that both DISP and WAF exposure induced a slight increase in the PCO levels but without significant differences compared with the Control (one-way ANOVA, p = 0.92 and 0.19 > 0.05 for DISP and WAF, respectively); while CEWAF exposure produced a significant elevation of the PCO level (one-way ANOVA, p = 0.04 < 0.05). As for lipid peroxidation, MDA contents were measured ( Figure 4D). Results revealed that the MDA contents in WAF and CEWAF treatments were much higher than that in the Control (one-way ANOVA, p < 0.01); while the MDA content in DISP treatment was subtly lower than that in the Control but without significant difference (one-way ANOVA, p = 0.67 > 0.05). Moreover, the MDA content in CEWAF treatment was also much higher than that in WAF treatment (one-way ANOVA, p < 0.001).  Control was the pre-filtered natural seawater only (black filled). Asterisks denote significant differences between the treatments and the Control. Dark traits denote significant differences among the treatments (one-way ANOVA).

IBR Index
IBR indexes indicated that CEWAF exposure caused greater variations in biomarker responses, with an average IBR index of 7.71, followed by WAF exposure with an average IBR index of 1.92 ( Figure 5). DISP exposure induced a slight variation in biomarker responses, with an average IBR index of 0.76, which was attributed to the changes in MDA content. IBR index results revealed that CEWAF exposure promoted more pronounced effects in biomarker responses related to the oxidative stress than DISP and WAF did. , water-accommodated fractions (WAF, dark-gray filled) and chemically enhanced WAF (CEWAF, light-gray filled) of Oman crude oil. The Control was the pre-filtered natural seawater only (black filled). Asterisks (*, **, or ***) denote significant differences between the treatments and the Control (p < 0.05, 0.01, or 0.001, respectively). Dark traits denote significant differences among the treatments (one-way ANOVA).

IBR Index
IBR indexes indicated that CEWAF exposure caused greater variations in biomarker responses, with an average IBR index of 7.71, followed by WAF exposure with an average IBR index of 1.92 ( Figure 5). DISP exposure induced a slight variation in biomarker responses, with an average IBR index of 0.76, which was attributed to the changes in MDA content. IBR index results revealed that CEWAF exposure promoted more pronounced effects in biomarker responses related to the oxidative stress than DISP and WAF did.  Control was the pre-filtered natural seawater only (black filled). Asterisks denote significant differences between the treatments and the Control. Dark traits denote significant differences among the treatments (one-way ANOVA).

IBR Index
IBR indexes indicated that CEWAF exposure caused greater variations in biomarker responses, with an average IBR index of 7.71, followed by WAF exposure with an average IBR index of 1.92 ( Figure 5). DISP exposure induced a slight variation in biomarker responses, with an average IBR index of 0.76, which was attributed to the changes in MDA content. IBR index results revealed that CEWAF exposure promoted more pronounced effects in biomarker responses related to the oxidative stress than DISP and WAF did.

Discussion
The use of chemical dispersants following an accidental oil spill could enhance the natural process (e.g., dissolution and microbial biodegradation) of petroleum hydrocarbons by reducing the interfacial tension between oil slicks and water, and mitigate the impact of oil slicks on the coastal habitats and human activities [44]. Nevertheless, chemical dispersion could also lead to the dissolution of more oil-derived hydrocarbons from oil droplets into the water column and then raise the threat of dispersed oil components to marine organisms, especially to marine benthos [24,45]. Sea cucumber, as a common member of marine benthic communities and an important marine aquaculture species, is mainly cultured in the coastal zone, where it is easily threatened by oil pollution. However, the impact of crude oil and chemical dispersant on sea cucumber is still not known. Hence, we exposed sea cucumber to DISP, WAF, and CEWAF solutions for 24 h, and measured the biochemical parameters related to oxidative stress responses to evaluate and compare the toxic effects of crude oil and chemically dispersed crude oil on sea cucumber.
Results manifested that ROS production increased in the respiratory tree of sea cucumber exposed to WAF, and the effect was much more obvious when exposed to CEWAF. The increase in ROS production observed in the present study is consistent with recent reports supporting that oil-derived hydrocarbons could elevate ROS production and the addition of chemical dispersant could enhance the elevation of ROS production in marine benthos, such as marine mussel (Mytilus galloprovincialis) [7] and eastern oyster (Crassostrea virginica) [24]. It is well known that the antioxidant enzymatic system is the primary defense line to scavenge the excessive ROS and maintain the cellular redox homeostasis within certain limits [46,47]. Consistently, T-AOC, as an important biomarker for assessing the antioxidant defense capacity, showed an increasing trend following WAF exposure in the present study. Differently, a significant reduction in T-AOC was observed following CEWAF exposure compared with the Control, indicating that CEWAF exposure could produce severe damage to the antioxidant defense system in the respiratory tree of sea cucumber. Previous studies have reported that exposure to oil-derived hydrocarbons at relative high concentrations could compromise the ability of antioxidant defense system in marine benthos, which exhibited a significant reduction in the antioxidant enzymatic activities, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [19,23,48], or in the T-AOC used in the present study, which is an integrated parameter to evaluate the cumulative action of all the antioxidants present in vivo [49]. The damage of the antioxidant defense system could lead to

Discussion
The use of chemical dispersants following an accidental oil spill could enhance the natural process (e.g., dissolution and microbial biodegradation) of petroleum hydrocarbons by reducing the interfacial tension between oil slicks and water, and mitigate the impact of oil slicks on the coastal habitats and human activities [44]. Nevertheless, chemical dispersion could also lead to the dissolution of more oil-derived hydrocarbons from oil droplets into the water column and then raise the threat of dispersed oil components to marine organisms, especially to marine benthos [24,45]. Sea cucumber, as a common member of marine benthic communities and an important marine aquaculture species, is mainly cultured in the coastal zone, where it is easily threatened by oil pollution. However, the impact of crude oil and chemical dispersant on sea cucumber is still less known. Hence, we exposed sea cucumber to DISP, WAF, and CEWAF solutions for 24 h, and measured the biochemical parameters related to oxidative stress responses to evaluate and compare the toxic effects of crude oil and chemically dispersed crude oil on sea cucumber.
Results manifested that ROS production increased in the respiratory tree of sea cucumber exposed to WAF, and the effect was much more obvious when exposed to CEWAF. The increase in ROS production observed in the present study is consistent with recent reports supporting that oil-derived hydrocarbons could elevate ROS production and the addition of chemical dispersant could enhance the elevation of ROS production in marine benthos, such as marine mussel (Mytilus galloprovincialis) [7] and eastern oyster (Crassostrea virginica) [24]. It is well known that the antioxidant enzymatic system is the primary defense line to scavenge the excessive ROS and maintain the cellular redox homeostasis within certain limits [46,47]. Consistently, T-AOC, as an important biomarker for assessing the antioxidant defense capacity, showed an increasing trend following WAF exposure in the present study. Differently, a significant reduction in T-AOC was observed following CEWAF exposure compared with the Control, indicating that CEWAF exposure could produce severe damage to the antioxidant defense system in the respiratory tree of sea cucumber. Previous studies have reported that exposure to oil-derived hydrocarbons at relative high concentrations could compromise the ability of antioxidant defense system in marine benthos, which exhibited a significant reduction in the antioxidant enzymatic activities, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [19,23,48], or in the T-AOC used in the present study, which is an integrated parameter to evaluate the cumulative action of all the antioxidants present in vivo [49]. The damage of the antioxidant defense system could lead to insufficient capacity for scavenging ROS and then a significant net increase in ROS levels, which subsequently could result in extensive oxidative damage of the macromolecules (DNA, proteins, and lipids) [50,51]. In the present study, we also evaluated the oxidative damage of these macromolecules in the respiratory tree of sea cucumber based on the levels of 8-OHdG, PCO, and MDA. We found that CEWAF exposure could produce a more significant increase in the PCO and MDA levels than did WAF exposure, which was accounted for 1.13-and 1.93-fold change, respectively. DISP exposure caused no apparent impact on the PCO and MDA levels. Besides, no increase in the level of oxidative DNA damage (8-OHdG) was observed in DISP, WAF, and CEWAF treatments. Our data suggested that chemically dispersed crude oil could enhance the oxidative damage in the respiratory tree of sea cucumber, which mainly manifested as protein oxidation and lipid peroxidation. Furthermore, the change in MDA level was much larger than the PCO level caused by WAF and CEWAF exposure, implying that lipid peroxidation could be the relatively more susceptible bioindicator of ROS-generated oxidative damage caused by oil-derived hydrocarbons exposure.
Several studies recently published on comparative analysis of toxic effects between WAF and CEWAF on various marine organisms have reported that chemical dispersant could increase the bioavailability of dissolved oil hydrocarbons and then enhance the toxicity of crude oil on marine organisms [9,10,52]. Our chemical analysis results manifested that both TPH content (around 13.60-fold) and PAHs level (around 2.52-fold) in CEWAF were higher than those in WAF. Moreover, compared with WAF, the proportion of HMW PAHs is much higher in CEWAF, especially in 3-ringed PAHs, which has been reported to be the predominant toxic components of oil-derived hydrocarbons to marine organisms [53,54]. It has also been documented that PAHs could elevate the ROS generation during their metabolism and biotransformation processes in marine benthos, leading to an imbalance between the ROS generation and scavenging [18,19,55,56]. Likewise, the ΣPAHs concentrations in WAF and CEWAF coincided with the oxidative damage observed in the present study. Consequently, our results supported these previous findings that the enhancement of the oxidative damage in marine benthos caused by oil-derived hydrocarbons could be attributed to the presence of chemical dispersants that solubilize more hydrocarbons from oil droplets into the water column, resulting in elevating their bioavailability to marine benthos.
Overall, the present study focused on comparative analysis of the antioxidant response and oxidative stress in the respiratory tree of sea cucumber following acute exposure to DISP, WAF and CEWAF. Our results revealed that chemical dispersant enhanced oxidative damage caused by crude oil to sea cucumber, which could be ascribed to its ability to solubilize higher levels of oil-derived hydrocarbons into the water column. Our data also suggested that the application of chemical dispersant in oil spill could increase the exposure of marine benthos to crude oil. Consistently, previous studies have reported that the application of chemical dispersant could also enhance the toxic effects of spilled oil to other marine organisms, such as plankton communities [57] and fishes [58]. Hence, comprehensive consideration of ecological effects of the application of chemical dispersant as an OSR is essential in OSR planning and decision making during accidental oil spills. Also, the potential health risk of oil and chemical dispersant to the respiratory system of humans, especially oil-cleaning rescue workers, should be considered before using the chemical dispersant [59]. Our findings on the oxidative responses associated with acute exposure to crude oil and chemically dispersed oil could provide a theoretical basis and guidance for the health management of sea cucumber culture. Future research should work on the investigation of the underlying mechanisms involved in oxidative stress response of sea cucumber to oil-derived hydrocarbons.