Effects of Strontium Marking on Otolith Elemental Deposition, Digestive Enzymes, and Antioxidant System in Juvenile Japanese Flounder (Paralichthys olivaceus)

Abstract

Japanese flounder (Paralichthys olivaceus) is an economically important species in China’s marine fishery industry. However, due to long-term intensive fishing, its wild population has declined sharply. Artificial stock enhancement has become a core measure for restoring its resources. This study aimed to investigate the effects of different immersion durations and concentrations of SrCl₂ solution (10, 20, 40, 80 mg/L) on strontium (Sr) deposition in the otoliths of P. olivaceus, and to systematically evaluate the impacts of Sr marking on the fish’s antioxidant capacity and digestive enzyme activity. The results showed that the otolith Sr/Ca ratio was positively correlated with marking concentration and duration; the optimal parameters were 40 mg/L for 4 days, with the Sr/Ca ratio returning to baseline after 30 days post-marking, and a 100% marking success rate. There were no significant differences in body length, body weight, or condition factor between the experimental groups and the control group (p > 0.05), but mortality was significantly increased in the 80 mg/L group. Digestive enzymes exhibited a dose-dependent response to Sr exposure, characterized by activation at low concentrations and inhibition at high concentrations; lipase was the most sensitive, with an inhibition threshold of 10 mg/L. Sr marking within the range of 20–40 mg/L for 4–8 days significantly activated the activity of T-AOC, CAT, GPx, and SOD (p < 0.05) and reduced MDA content, indicating that the antioxidant system was activated without causing persistent oxidative damage. In conclusion, Sr marking is a safe and efficient method for otolith marking in Paralichthys olivaceus. The recommended protocol is immersion in a 40 mg/L SrCl₂ solution for 4 days, followed by a 30-day recovery period in clean seawater before being used for stock enhancement evaluation. This study provides a scientific basis and technical support for assessing the effectiveness of stock enhancement in P. olivaceus.

1. Introduction

The Japanese flounder (Paralichthys olivaceus) is an economically important fish species in the Yellow Sea and Bohai Sea waters of China. Due to its tender meat and high economic value, it occupies a significant position in marine fisheries [1]. However, long-term intensive fishing combined with environmental changes in coastal waters has led to a marked decline in its wild populations [2,3]. According to statistics, in 2025 alone, Liaoning Province released over 1.4 million juvenile P. olivaceus with an average body length of more than 8 cm into the Yellow Sea in a single event, and the total release of the three major species—Chinese shrimp, swimming crab, and P. olivaceus—along the province’s coast reached 3.3 billion individuals, with an input–output ratio for stock enhancement exceeding 1:11. Thus, stock enhancement of P. olivaceus has become a core measure for restoring its wild populations and improving the coastal ecological environment.

The crux of assessing the efficacy of stock enhancement resides in precisely differentiating released individuals from wild ones. Conventional marking approaches encompass tag attachment, fin clipping, fluorescent dye marking, and coded wire tagging. Nevertheless, these methods generally encounter challenges such as high requirements for the body size of marked individuals, high operational mortality, and susceptibility to tag loss or fading, rendering them difficult to fulfill the practical requirements of large-scale release programs [4]. In recent years, strontium (Sr) marking technology based on otolith microchemistry has garnered extensive attention owing to its merits, including low costs, a high marking success rate, and a permanent retention of the mark signal. Otoliths are calcium carbonate crystals, formed through biomineralization in the inner ear of teleost fish. Once deposited, they are not reabsorbed by the fish, and the alterations in chemical elements from the core to the edge can comprehensively record the environmental history that the fish has experienced from birth to capture [5,6]. Sr²⁺ can enter the otolith lattice by substituting for Ca²⁺, forming a stable elemental fingerprint mark, a characteristic that makes it an ideal marking element [7].

Otolith microchemical analysis is the core tool for detecting strontium marks. This technique measures the content and distribution of elements such as Sr and Ca in otoliths by using electron probe microanalysis (EPMA) or laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) and uses the Sr/Ca ratio to reflect the fish’s habitat history and marking history [8,9]. Thomas et al. demonstrated that trace elements in otoliths are deposited through binding with proteins or calcium components, and that LA-ICP-MS enables high-resolution (<10 μm) microchemical imaging analysis, providing technical support for the precise identification of marked regions [6]. To date, Sr marking technology has been successfully applied to evaluate the effectiveness of stock enhancement in multiple fish species, i.e., Ctenopharyngodon idellus [10], Myxocyprinus asiaticus [11], Liza haematocheila [12], Takifugu obscurus [13], and Larimichthys crocea [14]. Zhu et al. immersed C. idellus larvae in 80 mg/L SrCl₂·6H₂O for 2 days and observed clear strontium marking peaks in all three types of otoliths; the Sr/Ca ratio gradually decreased to normal levels during the post-marking recovery period, and there was no significant difference in mortality between the control and experimental groups [10]. Yang et al. reported that in M. asiaticus, strontium residues in muscle could be completely metabolized within 7 days at concentrations of 12–18 mg/L, with no significant difference in mortality among concentration groups, demonstrating the safety and feasibility of this technique for release marking [11]. Zhang et al. (2024) further confirmed in juvenile L. haematocheila that immersion in 40 mg/L SrCl₂ for 1 day or 20 mg/L for 2 days produced stable marks in otoliths, with the mark signal still visible after 90 days of subsequent rearing, and no significant differences in body length, body weight, condition factor, or mortality between the marked and control groups [12].

As an exogenous metal element, Sr not only deposits in otoliths after entering the fish body but may also be distributed to other tissues and organs via blood circulation, potentially affecting the physiological functions of fish. Previous studies have shown that heavy metal exposure can disrupt the balance between reactive oxygen species (ROS) and the antioxidant defense system by inducing oxidative stress, leading to tissue damage [15,16]. In the antioxidant system, total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) are key enzymatic indicators, while malondialdehyde (MDA) is an important biomarker reflecting the degree of lipid peroxidation damage [16,17]. Xie et al. found that cadmium exposure induced significant increases in SOD, CAT, and GPx activity in the liver of Danio rerio, accompanied by a corresponding increase in MDA content, indicating that the antioxidant system was activated to scavenge excessive free radicals [16]. Similarly, Ma’rifah et al. confirmed in Nile tilapia that heavy metal exposure led to significant increases in SOD, CAT activity, and MDA levels, and that the enhancement of antioxidant enzyme activity could effectively scavenge ROS and prevent excessive accumulation of MDA [17].

Digestive enzyme activity is an important indicator, reflecting the digestive physiological function of fish and their response to environmental stress. Ecological factors such as temperature, salinity, pH, and heavy metal ions can all affect the activity of fish digestive enzymes [18,19,20]. Chen et al. found that under Cd²⁺ and Cu²⁺ stress, digestive enzyme activity in the lower concentration groups of Tegillarca granosa was enhanced in the early stage of the experiment but weakened in the later stage, while the higher concentration groups were inhibited from the beginning [19]. A similar conclusion was supported by Jiang et al. that Carassius carassius exposure to a high concentration of copper (0.60 mg/L) for 20 days significantly inhibited digestive and antioxidant enzyme activity; however, after 20 days of recovery, all enzyme activities returned to control levels [20]. These studies indicate that the effects of exogenous metal elements on a fish’s digestive function and antioxidant system are reversible, providing an important reference for the safety evaluation of strontium marking technology.

Despite these advances, research on the effects of strontium marking on digestive enzyme activity and the antioxidant system in fish remains very limited. Existing studies have mostly focused on the marking effect itself, i.e., whether a recognizable Sr/Ca peak is formed in the otolith, the marking success rate, and the persistence of the mark signal, while systematic assessments of the responses of fish digestive function and antioxidant defense systems during the marking process are lacking. Furthermore, as an important species for stock enhancement in China, the optimal parameters for otolith strontium marking and the evaluation of its physiological safety in P. olivaceus have not yet been systematically reported. Therefore, this study used juvenile P. olivaceus as the research object, set different SrCl₂·6H₂O concentrations (10, 20, 40, and 80 mg/L) and different immersion durations (2, 4, and 8 d), measured the otolith Sr/Ca ratio by LA-ICP-MS, and simultaneously determined the activity of pepsin, trypsin, α-amylase, and lipase, as well as antioxidant indicators, including T-AOC, SOD, CAT, GSH-Px, and MDA. We hypothesize that (1) an optimal SrCl2 concentration and immersion time exists that yields a 100% marking success rate without adversely affecting growth or survival; (2) Sr exposure induces a dose-dependent, biphasic response in digestive enzyme activity, characterized by activation at low concentrations and inhibition at high concentrations; and (3) the antioxidant system is activated by Sr exposure in a concentration- and time-dependent manner, and that this activation effectively prevents persistent oxidative damage. The aims were as follows: to optimize the optimal parameters for otolith strontium marking in P. olivaceus; to systematically evaluate the effects of strontium marking on digestive enzyme activity; and to elucidate the regulatory role of strontium marking on the antioxidant system. The results will provide safe and efficient technical parameters for strontium marking to evaluate the effectiveness of stock enhancement in P. olivaceus and offer a scientific basis for the physiological safety assessment of this technique.

2. Materials and Methods

2.1. Experimental Design

The P. olivaceus used in this study were obtained from Dalian Haolin Aquatic Products Co., Ltd. (Dalian, China). A total of 390 healthy individuals were selected. Prior to purchase, the fish had been cultured for approximately two months. The experiments were conducted in the Center for Marine Ranching Engineering Science Research of Liaoning. Before the experiments, aerated seawater (aerated for 2 days) was prepared in temporary holding tanks. Healthy fish showing no signs of disease were selected and placed in temporary holding tanks for 2 days of acclimation without feeding.

A total of 13 culture containers of identical volume and shape were each filled with 20 L of seawater. SrCl₂·6H₂O (molecular weight: 266.62, Shanghai Aladdin Biochemical Technology Co., Ltd. Shanghai, China) solutions were prepared at five different concentrations: 0 mg/L, 10 mg/L, 20 mg/L, 40 mg/L, and 80 mg/L. The 0 mg/L group served as the control, and the other concentrations served as experimental groups. P. olivaceus of similar size were randomly selected from the acclimation tanks and distributed into the 13 containers at a density of 30 fish per container, and the experiment was commenced simultaneously in all containers. All experimental fish were reared under identical environmental conditions: natural water temperature of 24 ± 1 °C, salinity maintained at 30 ± 1, and shade cloths were installed to prevent exposure to sunlight and light.

The experiment was divided into 3 phases. In the first phase, all samples were kept in tanks equipped with air pumps for 2 days without feeding. In the second phase, the fish were immersed in strontium-enriched seawater at the different concentrations described above. During the marking period, five fish were collected from each concentration group at marking durations of 2, 4, and 8 days, placed in individually labeled bags, and stored frozen. In the third phase, the experimental groups were transferred to normal seawater and cultured for an additional 30 days.

During the marking immersion period, the water was continuously aerated. Formulated feed was provided twice daily, and dead fish were promptly removed. Waste was cleaned, and water was exchanged once daily, with 1/2 of the water volume replaced. After the water exchange, SrCl₂·6H₂O was added proportionally to the experimental groups for which marking had not yet been completed in order to maintain stable Sr concentrations and a consistent water environment, thereby ensuring accurate experimental data.

2.2. Biological Measurement and Tissue Collection

The body length (accurate to 1 mm) and body weight (accurate to 0.01 g) of the experimental fish were measured. The condition factor (K) was calculated according to the formula [21]:

K = (W/L³) × 100,

(1)

where W is body weight (g), and L is body length (mm). After measurement, the sagittal otoliths were removed from the vestibular sacs of the fish, rinsed with clean water to remove surface impurities, placed in 2 mL centrifuge tubes, and preserved in 75% alcohol. Stomach tissue (for determination of pepsin, trypsin, lipase, and α-amylase) and liver tissue (for determination of T-AOC, CAT, GSH-Px, SOD, and MDA) were dissected and collected.

2.3. Otolith Preparation

The otoliths were placed into molds, embedded with prepared resin, and left to harden in a cool place. The hardened resin blocks were cut to an appropriate size, and the grinding surface was selected. The otoliths were ground close to the core region using sandpapers of grit sizes 600, 1200, and 2500 in sequence, and then polished with a 0.05 μm alumina suspension (Dongguan Songyoo Electronics Co., Ltd. Dongguan, China).

2.4. Trace Element Analysis

Laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS, Agilent 7700x, Agilent Technologies, Santa Clara, CA, USA) was used to perform spot analysis on the Sr-marked region (near the otolith edge). A line scan was conducted along the longest axis from the edge toward the core over a length of 400 μm at a scan speed of 0.6 μm/s in linear scanning mode. Helium was used as the carrier gas, and argon as the make-up gas to adjust sensitivity. The spot diameter was 40 μm, and two elements (⁴³Ca and ⁸⁸Sr) were analyzed at each ablation point. Quantitative analysis was performed using a multi-standard external calibration method, with USGS reference glasses (BIR-1G, MACS-3, NIST-610) used as calibration materials. The instrument operating parameters are listed in

Table 1. Analytical parameters of LA-ICP-MS.

Parameter	Value	Parameter	Value
Wavelength	150 nm	RF power	1350 W
Energy density	7.0 J/cm3	Plasma gas flow rate	15 L/min
Carrier gas	Helium (He, 0.65 L/min)	Auxiliary gas flow rate	1 L/min
Ablation spot diameter	40 μm	Make-up gas flow rate	0.7 L/min
Repetition rate	10 Hz	Sampling depth	5 nm
Ablation mode	Linear/constant speed scan	Detector mode	Dual

.

2.5. Enzyme Activity Assays

Stomach and liver tissue samples, collected according to concentration and time groups, were quickly weighed, recorded, and rinsed with pre-cooled physiological saline. Pre-cooled buffer was added at a certain weight-to-volume ratio, and the tissues were mechanically homogenized under ice-bath conditions. After centrifugation (4 °C, 3000–10,000× g, 10 min), the supernatant was collected as the crude enzyme extract, aliquoted, and stored at −80 °C for subsequent determination of enzyme activity and protein concentration. Pepsin, Trypsin, α-amylase, Lipase, T-AOC, CAT, GSH-Px, SOD, and MDA were analyzed by related kits according to corresponding instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.6. Data Processing and Statistical Analysis

The Sr content in otoliths is much lower than the Ca content. Following conventional practice, the Sr/Ca ratio is uniformly expressed as (Sr/Ca) × 10³ (mmol/mol), hereinafter referred to as the Sr/Ca ratio. The Sr/Ca ratios in different otolith regions are presented as mean ± standard deviation (mean ± SD). Offline data processing, including a selection of sample and blank signals, correction for instrumental sensitivity drift, and calculation of elemental concentrations, was performed using ICPS Data Cal software (version 10.9) [22].

Marking success was defined as the presence of a clearly identifiable Sr/Ca peak exceeding the baseline level (control range) in the LA-ICP-MS line-scan profile. The marking success rate was calculated as the percentage of otoliths displaying such a discernible Sr peak among all otoliths analyzed. All experimental fish immersed in Sr-enriched seawater were examined for the presence of a Sr mark; every individual showed a discernible Sr peak.

All statistical analyses were performed using SPSS 26 (version 26.0.0.0, IBM Corp., Armonk, NY, USA) [23]. Data are expressed as mean ± standard deviation (SD). For each dependent variable, a one-way ANOVA was conducted with a SrCl₂ concentration (0, 10, 20, 40, or 80 mg/L) as the between-subjects factor. When the omnibus F-test was significant, Tukey’s HSD post hoc test was applied to identify specific group differences; Tukey’s HSD controls the family-wise error rate across all pairwise comparisons within each dependent variable [24]. Exact p-values (rounded to three decimal places; p < 0.001 where applicable) and effect sizes as partial eta squared (η²p), interpreted following Cohen’s (1988) benchmarks, are reported for all analyses [25]. Graphs were prepared using Origin 2024 software [26].

3. Results

3.1. Effect of Sr Marking

The results of the P. olivaceus marking experiment showed that the Sr/Ca ratio in the otoliths of the control group remained relatively stable (2.99–4.02 mmol/mol), with no significant fluctuations during the experimental period and the subsequent rearing period. In contrast, the Sr/Ca ratio in the experimental groups varied considerably (2.77–6.46 mmol/mol). Complete Sr/Ca line profiles for all four SrCl₂ concentrations (10, 20, 40, and 80 mg/L) and all three immersion durations (2, 4, and 8 days) are presented in Figure 1 (marking groups) and Figure 2 (recovery groups). The Sr/Ca ratio was generally higher near the region approximately 1–1.5 mm from the core, after which it slightly decreased and stabilized at around 4 mmol/mol in the marked groups. The 40 mg/L marking concentration achieved a significant marking effect while maintaining low mortality, and the post-marking rearing phase was relatively stable; therefore, it was selected as the optimal marking concentration. Regarding marking duration, there was no statistically significant difference between the 4-day and 8-day treatments (p > 0.05). Based on operational efficiency, 4 days is recommended as the optimal marking duration (Figure 1 and Figure 3). Subsequently, after 30 days of recovery rearing, the Sr/Ca ratio returned to baseline levels (Figure 2 and Figure 3).

3.2. Effects of Sr on the Growth of P. olivaceus

Strontium marking had no significant effect on the growth performance of P. olivaceus (

Table 2. Body length and body weight of P. olivaceus affected by different strontium immersion marking concentrations and times, with one-way ANOVA results.

Marking Treatment Method			Body Length (mm)/Body Weight (g)				ANOVA
			10 mg/L	20 mg/L	40 mg/L	80 mg/L	F	p	η2p
Marking stage	Marking treatment group	2d	67.8 ± 8.75/ 1.81 ± 0.72	74.2 ± 6.87/ 2.37 ± 0.57	63.2 ± 8.34/ 54.0 ± 16.8	69.6 ± 5.5/ 1.97 ± 0.28	1.289/ 2.557	0.260/ 0.120	0.023/ 0.037
		4d	66.2 ± 6.34/ 1.89 ± 0.22	71.6 ± 7.19/ 2.19 ± 0.91	71.6 ± 6.26/ 2.54 ± 0.58	72.2 ± 6.53/ 2.64 ± 0.78
		8d	73.6 ± 6.76/ 2.58 ± 0.47	72.4 ± 6.8/ 2.36 ± 0.47	67.6 ± 6.18/ 1.95 ± 0.67	66.6 ± 3.84/ 2.03 ± 0.31
	Control group		72.25 ± 6.44/2.14 ± 0.59
Recovery stage	Marking treatment group	2d	68.3 ± 7.57/ 1.89 ± 0.37	77 ± 1.21/ 2.49 ± 0.22	75.1 ± 4.1/ 2.37 ± 0.28	77.3 ± 4.04/ 2.58 ± 0.49	1.673/ 2.099	0.140/ 0.101	0.043/ 0.059
		4d	65.6 ± 3.51/ 1.91 ± 0.20	80.6 ± 5.5/ 2.69 ± 0.41	74.6 ± 4.04/ 2.21 ± 0.19	75.2 ± 11.53/ 2.56 ± 1.14
		8d	74.33 ± 1.52/ 2.39 ± 0.27	70.1 ± 7.01/ 1.92 ± 0.19	72.6 ± 1.71/ 2.36 ± 0.82	68.3 ± 5.5/ 1.80 ± 0.11
	Control group		73 ± 9.01/2.4 ± 0.66

Note: Values are mean ± SD.

). There were no significant differences in body length or body weight between the experimental groups (p > 0.05) and the control group (p > 0.05), indicating that under the conditions of this experiment, strontium marking did not adversely affect fish growth. The condition factor results for the four experimental groups and the control group at different immersion concentrations are shown in

Table 3. Condition factor (K, ×10⁻³) of P. olivaceus affected by different strontium immersion marking concentrations, with one-way ANOVA results.

Marking Treatment Method			Condition Factor				ANOVA
			10 mg/L	20 mg/L	40 mg/L	80 mg/L	F	p	η2p
Marking stage	Marking treatment group	2d	0.66 ± 0.12	0.50 ± 0.11	0.48 ± 0.12	0.66 ± 0.21	1.586	0.134	0.027
		4d	0.57 ± 0.13	0.58 ± 0.13	0.60 ± 0.50	0.65 ± 0.17
		8d	0.50 ± 0.05	0.68 ± 0.01	0.68 ± 0.58	0.69 ± 0.14
	Control group		0.56 ± 0.1
Recovery stage	Marking treatment group	2d	0.59 ± 0.84	0.54 ± 0.16	0.56 ± 0.02	0.50 ± 0.11	1.568	0.116	0.031
		4d	0.55 ± 0.24	0.51 ± 0.63	0.59 ± 0.11	0.64 ± 0.02
		8d	0.68 ± 0.12	0.53 ± 0.59	0.58 ± 0.78	0.50 ± 0.21
	Control group		0.62 ± 0.13

Note: Values are mean ± SD.

. There was no obvious trend in condition factor, and no significant difference in condition factor was observed between the control group and the experimental groups (p > 0.05).

During the first 48 h of marking, mortality was relatively low. After 96 h of marking, mortality rates were 2.22% in both the 10 mg/L and 20 mg/L groups, 4.44% in the 40 mg/L group, and 8.88% in the 80 mg/L group. After 96 h, mortality rates in the 40 mg/L and 80 mg/L groups reached 7.77% and 12.2%, respectively. After 8 days of marking, mortality rates in all groups were low: 2.22% (20 mg/L), 1.1% (40 mg/L), and 2.2% (80 mg/L), with no mortality in the 10 mg/L group. Throughout this period, no mortality occurred in the control group (Figure 4).

3.3. Effects of Sr on Digestive Enzyme Activity

The effects of strontium marking on digestive enzyme activity in P. olivaceus were concentration- and time-dependent (Figure 5 and Figure 6). Enzyme activity in the high concentration group was substantially elevated compared to the control, with a large effect size (

Table 4. One-way ANOVA results for digestive enzyme activity of P. olivaceus during strontium marking at different concentrations.

Enzyme	Time	ANOVA Values
		Marking Period			Recovery Period
		F	p	η2p	F	p	η2p
Pepsin	2d	4.429	0.041	0.624	0.613	0.625	0.187
	4d	0.382	0.769	0.125	0.830	0.514	0.237
	8d	1.063	0.417	0.285	0.638	0.612	0.193
Trypsin	2d	2.833	0.106	0.515	1.784	0.228	0.401
	4d	0.789	0.533	0.228	1.640	0.256	0.381
	8d	0.639	0.611	0.193	0.318	0.812	0.107
α-Amylase	2d	2.440	0.139	0.478	1.455	0.298	0.353
	4d	1.303	0.339	0.328	2.948	0.098	0.525
	8d	2.389	0.143	0.474	0.887	0.488	0.250
Lipase	2d	0.306	0.821	0.103	0.563	0.654	0.174
	4d	1.635	0.257	0.380	0.533	0.672	0.167
	8d	0.881	0.491	0.248	2.665	0.119	0.500

). For pepsin: a low concentration (10 mg/L) significantly activated activity in the short term (2 d) (0.3386 ± 0.0156 U/mg prot, p < 0.05), while a high concentration (80 mg/L) showed no significant inhibitory trend. After 4 d and 8 d, enzyme activity in all concentration groups returned to control levels (0.2061–0.3413 U/mg prot), with no persistent effect. For trypsin: a high concentration (80 mg/L) significantly activated activity (reaching 1097.13 U/mg prot at 2 d), while medium and low concentrations (20, 40 mg/L) inhibited activity (752.23 and 799.90 U/mg prot, respectively). After a 2-day recovery period, activity in the medium- and low-concentration groups rebounded, while the 80 mg/L group remained high. Overall, trypsin showed activation at high concentrations and inhibition at medium-low concentrations, with unstable effects. For α-amylase: 80 mg/L inhibited activity in the short term (4 d) (271.57 U/mg prot) but activated it in the long term (8 d) (434.93 U/mg prot); 20 mg/L caused persistent inhibition at multiple time points; 10 mg/L and 40 mg/L showed short-term activation. For lipase: medium and low concentrations (20 mg/L, 40 mg/L) significantly activated activity (20 mg/L reached 638.13 U/mg prot at 4 d; 40 mg/L reached 656.07 U/mg prot at 8 d). 80 mg/L only showed short-term inhibition (4 d, 479.20 U/mg prot) but activity rebounded sharply at 8 d and during the recovery period.

3.4. Effects of Sr on Antioxidant Enzyme Activity

The effects of strontium marking on the antioxidant system of P. olivaceus were concentration- and time-dependent due to the high effect size (Figure 7 and Figure 8,

Table 5. One-way ANOVA results for antioxidant enzyme activity and MDA contents of P. olivaceus during strontium marking at different concentrations.

Enzyme	Time	ANOVA Values
		Marking Period			Recovery Period
		F	p	η2p	F	p	η2p
T-AOC	2d	0.423	0.742	0.137	4.720	0.035	0.639
	4d	1.039	0.426	0.280	1.126	0.395	0.297
	8d	0.372	0.776	0.122	0.448	0.726	0.144
CAT	2d	0.398	0.758	0.130	0.206	0.890	0.072
	4d	0.229	0.874	0.079	1.147	0.387	0.301
	8d	2.169	0.170	0.449	0.364	0.781	0.120
GHS-Px	2d	0.822	0.517	0.236	2.940	0.099	0.524
	4d	0.821	0.518	0.235	1.528	0.280	0.364
	8d	0.959	0.457	0.265	0.170	0.914	0.060
SOD	2d	5.260	0.027	0.664	0.096	0.960	0.035
	4d	1.657	0.252	0.383	0.800	0.528	0.231
	8d	0.507	0.688	0.160	6.751	0.014	0.717
MDA	2d	3.843	0.057	0.590	0.668	0.595	0.200
	4d	0.239	0.866	0.082	1.852	0.216	0.410
	8d	0.966	0.454	0.266	0.587	0.640

). Low concentration (10 mg/L) induced an increase in T-AOC (reaching 23.47 U/mg prot during recovery), while a medium-high concentration (40 mg/L) decreased it to the lowest level at 4 d (12.90 U/mg prot), indicating a compensatory protective response at a low concentration and depletion of antioxidant reserves at a medium-high concentration. CAT was stably activated at a low concentration (10 mg/L, 75.27 U/mg prot at 2 d), persistently inhibited at a medium concentration (20 mg/L, decreasing to 50.74 U/mg prot at 8 d), and fluctuated then recovered at a high concentration (80 mg/L). For GSH-Px, low concentrations (10, 20 mg/L) activated it, while high concentrations (80 mg/L) inhibited it, with levels remaining low during recovery, suggesting a tendency for persistent inhibition by high-concentration Sr. For SOD, a medium concentration (40 mg/L) showed short-term activation (178.57 U/mg prot at 2 d), medium-high concentrations caused inhibition at 4 d, and a high concentration showed activation at 8 d and during recovery. No concentration-dependent aggravation of lipid peroxidation was observed in any treatment group; the 40 mg/L group showed significantly decreased MDA content at 2 d and during the 4-day recovery period (5.65 and 5.44 U/mg prot, respectively) (Figure 9). Sr marking at appropriate concentrations (20–40 mg/L) and durations (4–8 d) activated the antioxidant system (increased T-AOC, CAT, GSH-Px, SOD) and decreased MDA without causing persistent oxidative damage. However, 80 mg/L may lead to sustained inhibition of GSH-Px.

4. Discussion

4.1. Otolith Sr/Ca Ratio as an Elemental Fingerprint for Marking Identification

Otoliths are important auditory and balance organs in fish, and the trace elements deposited in their calcium carbonate matrix during growth can reflect the chemical characteristics of the water environment experienced by the fish [27]. In this study, the Sr/Ca ratio in otoliths in the control group of P. olivaceus remained stable within the range of 2.99–4.02 mmol/mol, which is consistent with the background value of otoliths of juvenile P. olivaceus under natural seawater conditions [28]. In the experimental groups, a distinct elevation of the Sr/Ca ratio was observed at approximately 1–1.5 mm from the otolith core, indicating that exogenous strontium was effectively incorporated into the growing layer of the otolith during the marking period, forming a recognizable “elemental fingerprint”. After the marking period, the Sr/Ca ratio rapidly decreased and returned to a level comparable to that of the control group. This dynamic change further confirms the capability of otolith microchemistry to record real-time changes in the water environment [29]. Therefore, the otolith Sr/Ca ratio can serve as a reliable natural marker for identifying the origin of fish populations or recognizing released individuals.

4.2. Effects of Strontium Marking on Growth and Survival

Growth performance and survival rate are key indicators for evaluating the safety of marking methods. In this study, no significant differences were observed in body length, body weight, or condition factor between any experimental group and the control group (p > 0.05), indicating that exogenous strontium marking did not significantly inhibit the growth of juvenile P. olivaceus. This is consistent with previous findings on the effects of strontium marking on fish growth [30,31,32]. However, a clear dose–effect relationship was observed between mortality and marking concentration. During the early marking period (within 96 h), the 80 mg/L group showed the highest mortality (8.88%), while the 10 mg/L and 20 mg/L groups exhibited lower mortality (2.22%). After 8 days of marking, cumulative mortality further increased in all groups, but the differences among groups were not significant (p > 0.05), suggesting that acute toxicity mainly occurred in the early stage of marking, possibly related to short-term osmoregulatory stress induced by high strontium concentrations [33]. The 40 mg/L group had a cumulative mortality of 4.44% after 96 h of marking, significantly lower than that of the 80 mg/L group, and no continuous mortality was observed during the subsequent recovery period, indicating that this concentration achieves a good balance between safety and marking effectiveness.

4.3. Effects of Strontium Marking on Digestive Enzyme Activity

Digestive enzyme activity is an important indicator reflecting the digestive physiological function of fish and their response to environmental stress. The present study found that the 40 mg/L Sr²⁺ group showed no significant difference from the control group in most enzymatic indicators, indicating that this is a relatively safe marking concentration. This finding is consistent with previous studies [11,12,34,35]. Yang et al. [11] reported that in M. asiaticus, no significant difference in mortality was observed within the Sr concentration range of 12–72 mg/L, and that strontium residues in muscle could be completely metabolized within 7 days at concentrations of 12–18 mg/L. Zhang et al. [12] also confirmed that growth and mortality in the marked groups did not differ from those in the control group in L. haematocheila. However, in the present study, 20 mg/L Sr²⁺ inhibited α-amylase and trypsin, whereas 80 mg/L Sr²⁺ activated them. This suggests that the response of digestive enzymes to Sr stress is non-linear and may involve concentration-dependent differences in the competition between Sr²⁺ and Ca²⁺ for binding sites. Song et al. [36] found that elevated Sr²⁺ inhibits the activity of Ca²⁺-ATPase and Na⁺/K⁺-ATPase, providing direct evidence that strontium interferes with calcium metabolism.

The enrichment and metabolic patterns of strontium have important implications for its physiological effects. Bo et al. [35] found that the strontium accumulation in various tissues of D. rerio followed the order of bone > liver > whole fish, while the excretion rate followed the order of whole fish > liver > bone, explaining why otoliths are ideal target organs for strontium marking. Klaczek et al. [37] demonstrated that aqueous strontium accumulates in the plasma and gills of Oncorhynchus mykiss in a dose-dependent manner, and significantly inhibits gill Ca²⁺-ATPase activity only at the highest concentration (1948 μM). Under conditions of high strontium and low calcium, the expression of liver calcium signaling receptor genes was suppressed, suggesting that strontium toxicity may act through interference with calcium signaling pathways, and that a high-calcium environment can protect fish from the acute effects of strontium exposure.

The present study found that digestive enzyme activity generally recovered during the recovery phase. In the 2-day treatment group, most enzyme activities returned to control levels, and similar recovery patterns were observed in the 4-day and 8-day groups. Zhang et al. [12] confirmed that after 36–38 days of post-marking rearing, strontium content in muscle returned to control levels, indicating that the effects of strontium marking are reversible. Enzyme activity fluctuations were most pronounced in the 2-day treatment group, while the 4-day and 8-day groups tended to be more stable, suggesting that extending the marking duration did not exacerbate abnormal fluctuations. Considering that Yang et al. [11] recommended a 7-day marking period and Zhang et al. [12] recommended 1–2 days for mullet, the present study suggests that a 4-day marking period allows fish to adequately adapt to the strontium environment while ensuring marking effectiveness, thereby reducing stress responses.

It should be noted that the digestive enzyme and antioxidant data for the 80 mg/L group were obtained exclusively from surviving individuals, as spontaneously deceased fish were removed promptly and not subjected to biochemical analysis to avoid post-mortem artifacts. This introduces potential survivorship bias, wherein the observed enzyme activity and antioxidant responses may underestimate the true physiological impact of high-concentration Sr exposure. The increased mortality in the 80 mg/L group itself constitutes direct evidence of severe physiological stress. Therefore, the apparently moderate biochemical responses in surviving fish from this group should not be interpreted as evidence of safety, but rather as reflecting the physiological characteristics of the subset that managed to withstand the treatment. The 80 mg/L concentration is therefore not recommended as a safe marking concentration.

4.4. Effects of Strontium Marking on Antioxidant Enzyme Activity

The antioxidant system is the first line of defense against oxidative stress in organisms. Among its key enzymatic indicators are AOC, SOD, CAT, and GSH-Px, while MDA reflects the degree of lipid peroxidation damage [38,39]. The present study showed that exogenous Sr²⁺ exposure induces oxidative stress in P. olivaceus, with response patterns being concentration- and time-dependent [40,41]. A low concentration induced an increase in T-AOC, while medium-high concentrations caused inhibition, consistent with the study by Cheng et al. [42] on Mytilus edulis. The increase in T-AOC at a low concentration represents a compensatory protective response to an initial oxidative threat, whereas the decrease at medium-high concentrations indicates depletion of antioxidant reserves [41,43]. During the recovery phase, T-AOC in all concentration groups returned to control levels, and some groups even showed values higher than the control, indicating good recovery ability. In the 8-day treatment group, CAT activity in the 20 mg/L group decreased to the lowest value overall (50.74 U/mg prot), showing a significant inhibitory effect. This non-monotonic dose effect is consistent with the findings of Cao et al. [44] and may involve competitive binding of Sr²⁺ to metal ions in the active center of CAT [45]. During the recovery phase, GSH-Px activity in all 80 mg/L treatment groups was lower than that of the control and remained at low levels, indicating a tendency for persistent inhibition by high-concentration Sr²⁺, consistent with the results of Cheng et al. [42]. No treatment group showed a trend of significant increase in MDA content with increasing Sr concentration; some groups (e.g., 40 mg/L 2-day treatment group, 40 mg/L 4-day recovery group) even exhibited lower MDA than the control. This indicates that Sr²⁺ exposure did not cause severe lipid peroxidation damage, and even high concentrations did not induce persistent oxidative damage, confirming the effective scavenging capacity of the antioxidant system [42]. SOD activity showed activation at medium concentrations (40 mg/L) and fluctuation at high concentrations, with a temporal dynamic of “activation-inhibition-reactivation”. This pattern echoes the “preparation for oxidative stress” (POS) strategy described by Cao et al. [44], reflecting the dynamic adaptive regulation of P. olivaceus to Sr stress [40,41]. Integrating the five indicators, the 40 mg/L Sr²⁺ concentration had the least impact on the antioxidant system of P. olivaceus, showing no significant difference from the control, or only moderate activation at most time points, consistent with the relatively safe concentration determined from digestive enzyme studies [34]. Studies by Yang et al. [11] on Chinese sucker also support this concentration range (12–18 mg/L Sr, with muscle residues completely metabolized within 7 days and no significant difference in mortality). Cheng et al. [42] further confirmed that Sr exposure can lead to increased ROS and DNA damage, providing a toxicological basis for the safe application of strontium marking [34,46,47].

5. Conclusions

This study established an optimal strontium marking protocol for juvenile P. olivaceus: immersion in 40 mg/L SrCl₂·6H₂O for 4 days, which achieved a 100% marking success rate with a clear and stable Sr peak in otoliths. The marker signal returned to baseline after 30 days of recovery in clean seawater. Strontium marking did not affect growth performance; however, the 80 mg/L concentration increased mortality and is not recommended. Digestive enzyme responses to Sr exposure were concentration-dependent and reversible, with all indicators returning to normal during recovery. Moderate Sr concentrations (20–40 mg/L) activated the antioxidant system without causing persistent oxidative damage, whereas 80 mg/L induced sustained GSH-Px inhibition. Overall, immersion in 40 mg/L SrCl₂·6H₂O for 4 days, followed by a 30-day recovery period in natural seawater, is a safe and effective method for marking juvenile P. olivaceus prior to release for stock enhancement evaluation.