Physiological Effects of Suspended Solids on Venerupis philippinarum and Argopecten irradians

Abstract

Suspended solids are small particles transported in the water column, which can damage marine ecosystems and impair the health of aquatic organisms. This study evaluated the physiological responses of clams (Venerupis philippinarum) and Atlantic Bay scallops (Argopecten irradians) to suspended solid exposure. Four concentrations (100–1000 mg/L) were tested, with a control group maintained at 0 mg/L. At each time point (1, 2, 4, 6, 8, and 12 days), hemolymph samples were collected from five individuals per group to measure GOT, GPT, ALP, and cortisol. Exposure to suspended solids significantly increased these biochemical indicators compared with the control. Quantitative survival analysis showed that Venerupis philippinarum survival declined to 83.3% (25/30) at 500 mg/L and 76.7% (23/30) at 1000 mg/L after 5 days, while the control maintained 100% survival. In Argopecten irradians, survival remained close to 100% in most treatments, with a slight reduction to 83.3% (25/30) at 1000 mg/L. No mortality occurred in the control group without suspended solids, whereas mortality was evident under combined temperature stress and suspended solid exposure. These findings demonstrate that suspended solids induce stress responses in both species, with early mortality in Venerupis philippinarum likely caused by particle adhesion to the gills, leading to reduced respiratory efficiency.

1. Introduction

Anthropogenic activities, such as harbor maintenance, construction, and development, cause serious problems in the marine environment, including changes in seafloor topography and destruction of spawning grounds. Further, suspended solids are known to cause respiratory distress and affect the health of marine organisms [1,2]. Suspended solids are small particles that are transported through the water column and cause damage to ecosystems and some fisheries [3]. They are generally composed of inorganic materials such as silt, clay, and sand, as well as organic matter including detritus, plankton, and fecal particles. In bivalve aquaculture areas, suspended solids can originate from natural processes such as tidal currents, storms, and river inflow, but they are also strongly influenced by anthropogenic activities including dredging, coastal construction, and resuspension caused by high stocking density and feeding practices [4]. Several jurisdictions publish numeric or narrative standards for total suspended solids (TSSs) to protect aquatic ecosystems. For example, British Columbia (Canada) limits human-induced TSS to ≤10 mg/L when the background is ≤100 mg/L, and to ≤10% of background when it exceeds 100 mg/L [5]. Minnesota (USA) sets a summer-average TSS standard of 30 mg/L for Class 2B waters [6]. For coastal/mariculture waters, the South African guideline (drawing on ANZECC/ANZG methods) recommends site-specific values not exceeding the 80th percentile of reference conditions and cites <10 mg/L for marine waters as a trigger value used in other jurisdictions [7]. Within the EU (including the Netherlands), the Water Framework Directive generally does not prescribe a single national TSS number; instead, member states set site-specific turbidity/TSS objectives and permit conditions derived from reference conditions [8,9]. However, they do not provide impact concentration levels, and scientific and objective assessments are required to establish appropriate standards for marine life. Suspended solids have been studied ecologically since the 1930s, starting with field experiments [10,11]. In the 1970s, rapid industrial development led to the conduct of bioassay studies on the effects of dredging and coastal erosion [12]. Until now, studies on the impact of suspended solids using fish are dominant (70%), while those using bivalves are relatively underwhelming (18%) [13]. As suspended solid concentration increases, invertebrates experience growth impairment due to changes in their prey biota as suspended solid concentrations increase. Furthermore, sustained suspended solid exposure can cause community changes owing to lower organismal tolerance and reduced species diversity [14,15]. Previous studies have investigated the physiological effects of suspended solids exposure on marine organisms, including surf clams (Mactra veneriformis), Asian hard clams (Meretrix lusoria), Yesso scallops (Patinopecten yessoensis), bloody clams (Scapharca broughtonii), and clams (Ruditapes philippinarum). A suspended solid concentration range of 100–4000 mg/L physically affects these organisms, and it causes bivalve mortality at 100–1000 mg/L [13].

The impact of suspended solids on marine organisms is of great importance as it advances our understanding of the environment and ecosystems and has important implications for sustainable marine conservation and ecosystem management. Suspended solids can reduce light penetration and photosynthesis, alter nutrient cycling, and impair the feeding efficiency of filter-feeding bivalves. Prolonged exposure has been shown to cause increased metabolic stress, reduced growth, impaired reproduction, and immune suppression, which may ultimately affect population dynamics and ecosystem stability. These effects underline the necessity of regulating suspended solid concentrations to safeguard aquaculture productivity and biodiversity conservation [15,16,17].

Clams are a species commonly distributed in marine ecosystems in various coastal regions of the Atlantic, Pacific, and Indian Oceans. However, their production has been declining due to habitat loss from landfill development [18]. The Atlantic Bay scallop (Argopecten irradians) is native to the North American Atlantic Ocean and was transplanted from North America to China in 1986, where it was mass-cultured. Owing to its rapid growth, this scallop was introduced to Korea through China and is cultivated in Tongyeong, Goseong, and Gyeongsangnam-do. Therefore, in this study, we aimed to determine the physiological effects of different suspended solid concentrations on clams (Venerupis philippinarum) and Atlantic Bay scallops (A. irradians), which are the main commercial species in Korea, via serological analyses. Water temperature stress was applied to confirm the immune decline after exposure to suspended solids.

2. Materials and Methods

2.1. Preparation of Experimental Organisms

The V. philippinarum and A. irradians used in this experiment were provided by a fish farm in Tongyeong, Gyeongsangnam-do, Republic of Korea. The V. philippinarum had an average length and height of 42.8 ± 1.2 mm and 20.6 ± 3.4 mm, respectively, and those of A. irradians were 62.7 ± 3.7 mm and 27.2 ± 4.3 mm, respectively. All samples were kept in a 400 L aquarium at 18 °C and dewatered by 50% twice daily until the start of the experiment.

2.2. Suspended Solids Exposure

To determine the effects of suspended solids on V. philippinarum and A. irradians, four suspended solid concentrations were selected, and a control group (0 mg/L) was set up. The suspended solids used in the experiment were silt collected from the coastal waters near Busan New Port (35°04′ N, 128°49′ E), which were passed through a 63 μm mesh; only the fraction that passed through was used in the bioassay experiment. Each treatment was conducted in 200 L fiberglass tanks, and natural seawater pumped directly from the adjacent coast was used throughout the experiment. An underwater motor was attached to each tank to keep the particles in suspension. The concentration of the suspended solids was adjusted to 100, 250, 500, and 1000 mg/L. To verify the concentration in each treatment, nephelometric turbidity units (NTUs) were measured using a turbidimeter (HANNA Instruments, HI98703, Woonsocket, RI, USA) with 10 mL seawater samples collected from each tank (

Table 1. The measurement of suspended solids exposure concentration was expressed in mg/L and nephelometric turbidity units (NTUs) for each experimental segment.

Group	Suspended Solids (mg/L)	Nephelometric (NTU)
Control	0	0
G1	100	55.2
G2	250	158
G3	500	413
G4	1000	718

). Each treatment, including the control, was conducted in three replicate tanks. The exposure experiment was conducted for 12 days at 18 °C. Each tank was stocked with 40 V. philippinarum and 40 A. irradians. Tanks were supplied with aeration to maintain dissolved oxygen levels above 6 mg/L, and 50% of the water was renewed daily, with suspended solids replenished to maintain the target concentrations. Salinity was maintained at 30 ± 1 PSU throughout the experiment. The collected suspended solids mainly consisted of inorganic minerals (silt and clay particles), organic detritus, and minor planktonic material; no chemical contaminants were detected in preliminary analyses. No food was supplied to the bivalves during the experimental period.

2.3. Serum Analysis

Serum samples were collected from five selected individuals at each sampling time point (days 1, 2, 4, 6, 8, 10, and 12). To avoid potential health effects and bias from repeated sampling, the same individuals were not used for successive collections; instead, each clam or scallop was sampled only once and then excluded from further analysis. Samples were kept at 4 °C overnight to clot, centrifuged at 3500 rpm for 10 min at 4 °C, and the separated serum was analyzed using a dry biochemistry analyzer (FUJI DRI-CHEM 4000i; Honshu, Japan).

2.4. Cortisol Analysis

To determine the stress hormone changes caused by suspended solids in V. philippinarum and A. irradians, cortisol levels were analyzed using an Oxford Biomedical Research cortisol kit (Oxford Biomedical Research, Rochester Hills, MI, USA). Briefly, 1 mL of ethyl ether was added to 100 $\mathsf{\mu }$L of prepared serum, vortexed, and reacted at −81 °C for 15 min. Only the supernatant was transferred to a new microtube and reacted in a vacuum concentrator until all the liquid has evaporated. Subsequently, 100 $\mathsf{\mu }$L of extraction buffer was added to the microtube, and then 10 $\mathsf{\mu }$L of the diluted solution was dispensed into a new microtube with 990 $\mathsf{\mu }$L of extraction buffer and vortexed. Then, 8 standards and the samples (10 $\mathsf{\mu }$L of dilution solution + 990 $\mathsf{\mu }$L of extraction buffer) were dispensed into a 96 well plate, 50 $\mathsf{\mu }$L of conjugate reagent was added, and the reaction was carried out for 1 h at room temperature. After the reaction, the plate was washed three times with 200 $\mathsf{\mu }$L of wash buffer, after which the TMB substrate was added in 150 $\mathsf{\mu }$L increments. The mixture was allowed to react for 30 min at room temperature, and the absorbance was measured at 450 nm after adding 1 N HCl.

2.5. Water Temperature Stress

To determine the survival rate of V. philippinarum and A. irradians after exposure to suspended solids, 30 individuals each of V. philippinarum and A. irradians were distributed in the suspended solids exposure tank, acclimatized for 1 week, and then warmed by 1 °C per day up to a maximum of 23 °C, with mortalities checked daily.

2.6. Statistical Analysis

All experiments were performed in triplicate for accuracy, and all data were expressed as mean and standard deviation. Significant difference between groups was determined using ANOVA with the SPSS statistical program (SPSS Inc. ver 20, Chicago, IL, USA). p < 0.05 was considered significant using Duncan’s multiple range test as a post hoc test.

3. Results

3.1. Cumulative Survival During Suspended Solids

V. philippinarum mortality following suspended solid exposure was 0 in the control, 100 mg/L, and 1000 mg/L. A mortality at 250 mg/L was recorded on day 4 following exposure, and two mortalities at 500 mg/L on day 6. Meanwhile, no mortality in the control tank was recorded for the A. irradians, one on day 2 in the 100 and 500 mg/L, and one on days 3 and 4 in the 250 mg/L tank. Finally, at the highest concentration of 1000 mg/L, one mortality was recorded on day 2, followed by an additional mortality on day 6.

3.2. Serum Analysis

GOT, GPT, and ALP levels were upregulated in the suspended solids group of A. irradians. In V. philippinarum, and no significant difference in GOT levels was observed between the control (0 mg/L) and suspended solids groups until day 4. However, on day 6, GOT was significantly upregulated in the 250 mg/L tank of the control group (0 mg/L), and on day 8, it was upregulated in the 100 mg/L tank. Further, no significant difference between the control and suspended solids groups of V. philippinarum was observed until day 2. On day 4, GOT levels were significantly upregulated in the 250 mg/L tank of the control group (0 mg/L); on day 6, it was significantly upregulated between 250 and 500 mg/L, and on day 8, it was significantly upregulated at 100 mg/L. Finally, V. philippinarum ALP was significantly downregulated in the control (0 mg/L) at 100 and 250 mg/L on day 1, followed by no significant difference, significant upregulation at 250 and 500 mg/L on day 6, and significant upregulation at 1000 mg/L on day 10 (Figure 1).

Meanwhile, analysis of the A. irradians showed a significant upregulation of GOT at 1000 mg/L compared to the control group (0 mg/L) from day 1 to day 4, followed by significant upregulation in the 100, 250, and 1000 mg/L groups on day 6 (Figure 2). On day 10, it was significantly upregulated at 100 and 500 mg/L and significantly downregulated at 250 mg/L (Figure 2). GPT levels were also significantly upregulated at 250, 500, and 1000 mg/L in the control group (0 mg/L) on day 1, followed by significant downregulation at 500 mg/L on day 8, and 100 mg/L and 1000 mg/L on day 10 (Figure 2). On day 12, it was significantly downregulated at 250 mg/L and upregulated at 500 mg/L. Finally, ALP was significantly upregulated at 1000 mg/L relative to the control group, significantly upregulated at 250 mg/L on day 4, and significantly downregulated at 100 mg/L on day 10 (Figure 2).

3.3. Cortisol Analysis

V. philippinarum cortisol was significantly upregulated in all suspended solids compared to the control group (0 mg/L) on day 1 and was significantly upregulated in the high-concentration tanks of 500 and 1000 mg/L on days 4 and 8. It was significantly upregulated at a concentration of 100 mg/L on day 12 (Figure 3). Meanwhile, A. irradians cortisol was significantly upregulated at 250, 500, and 1000 mg/L in the control group (0 mg/L) on day 1, 250 and 1000 mg/L on day 2, and significantly upregulated at 500 and 1000 mg/L on day 4. On day 6, it was upregulated at 500 mg/L relative to the control, and on day 8, it was significantly upregulated in both suspended solids concentration tanks. It was then significantly upregulated at 100 and 1000 mg/L on day 10, and finally at 250 mg/L on day 12 (Figure 3).

3.4. Cumulative Survival After Water Temperature Stress Stimulation

Mortality was determined after water temperature stress stimulation to determine the sensitivity of V. philippinarum to external environmental conditions following exposure to suspended solids. No mortality was observed with the V. philippinarum at control and 100 mg/L until day 5 (23 °C), when the experiment ended. However, mortality was observed at 250, 500, and 1000 mg/L. At 250 mg/L, one mortality was recorded on day 1 (19 °C), two mortalities on day 2 (20 °C), and one mortality on day 3 (21 °C); at 500 mg/L, two mortalities were recorded on days 1 and 2, and one mortality on day 4. Finally, 1000 mg/L resulted in one mortality each on days 1 and 3. In A. irradians scallops, no mortalities were recorded in the control and 250 mg/L groups until day 5, when the experiment ended. At 100 mg/L, one mortality was recorded each on days 1 and 2, and at 500 mg/L, one mortality was recorded on day 1. Finally, at 1000 mg/L, one mortality was recorded on day 1, three mortalities on day 2, and one mortality on day 4 (

Table 2. The survival of V. philippinarum and A. irradians was assessed following one week of exposure to suspended solids and subsequent water temperature stress stimulation.

		Survival Number/Total Number of Species
Species	Group	1D (19 °C)	2D (20 °C)	3D (21 °C)	4D (22 °C)	5D (23 °C)
V. philippinarum	Control	30/30	30/30	30/30	30/30	30/30
	100 mg/L	30/30	30/30	30/30	30/30	30/30
	250 mg/L	29/30	27/30	26/30	26/30	26/30
	500 mg/L	28/30	26/30	26/30	25/30	25/30
	1000 mg/L	29/30	29/30	28/30	28/30	28/30
A. irradians	Control	30/30	30/30	30/30	30/30	30/30
	100 mg/L	29/30	28/30	28/30	28/30	28/30
	250 mg/L	30/30	30/30	30/30	30/30	30/30
	500 mg/L	29/30	29/30	29/30	29/30	29/30
	1000 mg/L	29/30	26/30	26/30	25/30	25/30

).

Correlation analysis revealed significant or near-significant negative relationships between survival rate and water temperature at higher suspended solid concentrations (Figure 4). In V. philippinarum, the survival rate showed a significant negative correlation at 500 mg/L (r = –0.90, p < 0.05) and strong negative but borderline associations at 250 mg/L (r = –0.85, p = 0.069) and 1000 mg/L (r = –0.87, p = 0.058). In A. irradians, a negative correlation was also observed at 1000 mg/L (r = –0.87, p = 0.058), although this did not reach statistical significance.

4. Discussion

Unlike fish farming, shellfish farming is completely dependent on the marine ecosystem around the farm, with minimal management after the seedlings are attached to the water surface or sprayed onto the seabed [19]. Therefore, in this study, we exposed V. philippinarum and A. irradians to specific concentrations of suspended solids to determine their physiological effects and performed biochemical analyses to determine the effects of simultaneous suspended solids and water temperature stress.

A concentration range of 10–4000 mg/L of suspended solids is known to affect benthic organisms in Korea. Among bivalves, the surf clam, Asian hard clam, and Yesso scallop were found to be affected at a concentration range of 25–1000 mg/L [13]. In particular, bivalve lethality due to suspended solids has been reported to occur at 100–1000 mg/L [19]; therefore, in this study, we also conducted experiments with a suspended solid concentration range of 100–1000 mg/L.

In this study, after exposure to suspended solids, both V. philippinarum and A. irradians survived in the control group (0 mg/L), and mortality was observed only at the experimental sites. Mortality was observed in V. philippinarum at 250 and 500 mg/L and in A. irradians at all experimental sites. This difference was likely due to the morphological differences between organisms. Unlike scallops, V. philippinarum have inlet and outlet tubes that draw seawater into their bodies, filter out organic matter to feed on small microorganisms and plankton, and return seawater to the outlet tube. Bivalves, including both clams and scallops, possess incurrent and excurrent siphons that allow them to filter water. However, the siphons of scallops are relatively short and less developed compared to those of V. philippinarum [20]. In V. philippinarum, the siphons function in feeding under natural conditions but may also have acted as effective filters to reduce the intake of suspended solids in this experiment, which could explain the lower mortality observed in this species.

Furthermore, the difference in gill size between V. philippinarum and A. irradians may also be relevant, as gills are an important organ for respiration. In bivalves, the gills are the primary organ that responds to changes in the external environment, as they provide a large surface area in direct contact with the surrounding water. Previous studies have shown that fine sediment particles can adhere to the gills of bivalves, leading to respiratory impairment; when the gills become clogged, feeding is inhibited, and mortality may ultimately occur [21,22,23]. Therefore, in this experiment, it is likely that the gills of A. irradians, which are relatively large, had more contact with the seawater to which the suspended solids fish were exposed, and may have caused mortality.

In the present study, we found that the serum GOT, GPT and ALP levels were upregulated in V. philippinarum and A. irradians scallops after exposure to suspended solids. GOT, GPT, and ALP are serum markers that can detoxify or offset stressors as they enter or leave the body and are known to be active for a period of time. The increased activity of GOT and GPT in the hemolymph components of shellfish is usually caused by the entry of cells isolated from damaged tissues caused by environmental pollutants [24]. In addition, GOT and GPT are enzymes that catalyze amino acid group transfer reactions between amino acids and α-keto acids, are widely distributed in the body, and are used as indicators of histological damage [25]. Meanwhile, ALP catalyzes the hydrolysis of phosphate esters in vertebrates, promotes the mineralization of cartilage and bone, activates calcification, and plays an important role in the formation of new bone. They are also closely related to calcium, phosphate, and magnesium ions [26]. In marine invertebrates, ALP, also called AKP, is a glycoprotein present in the hemolymph and is an important lysosomal enzyme with non-specific immune functions, such as digestion or phagocytosis of foreign entities [27]. Previous studies have shown that higher concentrations of cadmium, a non-floating environmental pollutant, increased the concentration of GOT and GPT in oyster hemolymph, as well as decreased the expression of HSP90 and Mn-SOD mRNA. These results confirm the consequences of tissue damage caused by excessive stress [28]. Furthermore, ALP was significantly increased in northern abalone (Haliotis discus hannai) exposed to 28 °C [29]. Therefore, in the present study, GOT, GPT, and ALP were used as indicators to identify physiological changes in tissue damage caused by suspended solids, and our results are consistent with those of previous studies [30,31].

Fish exposed to stress generally exhibit three responses. First, there is an increase in the activity of the hypothalamic–sympathetic–chromaffin cell axis and the hypothalamic–pituitary–adrenal gland axis, which release catecholamines and cortisol, respectively, into the blood [32,33,34]. Secondary effects of catecholamines and cortisol in the blood and tissues include increased respiration rate, oxygen consumption, energy mobilization, and disruption of water-ion equilibrium [35,36,37,38]. In a previous study, we found that high-density stress significantly upregulated cortisol levels in mussel larvae [39]. When oysters were subjected to high-temperature stress, cortisol was significantly upregulated after 6 h, indicating that cortisol might be involved in the regulation of energy demand during the early response to high-temperature stress in oysters [40]. In this study, similar to previous studies, we were able to identify increased cortisol concentrations in V. philippinarum and A. irradians upon exposure to suspended solids, suggesting that suspended solids stimulate their nerves to release stress hormones.

Shellfish are affected by external environmental factors such as water temperature and salinity. When these environmental factors are unstable, productivity is reduced due to an energy imbalance for metabolism through the induction of biochemical and physiological changes, and temperature is a direct factor affecting metabolism and energy balance [41]. In this study, by subjecting experimental tanks to water temperature stress after exposure to suspended solids, we determined how another stressful stimulus adversely affects V. philippinarum and A. irradians that are already exposed to suspended solids. Results showed that no mortality occurred in the control group (0 mg/L) and in plots with relatively low concentrations. This suggests that, in stressful environments, suspended solids rapidly degrade the health of V. philippinarum and A. irradians.

In this study, we conducted serum and cortisol analyses of the hemolymph of V. philippinarum and A. irradians to determine the physiological effects of suspended solids, as well as their survival after water temperature stress. All the experimental data confirm that exposure to suspended solids had a negative effect on V. philippinarum and A. irradians. Although we did not perform tissue analysis to confirm tissue damage in this study, we were able to infer tissue damage by examining biological indicators. However, the extent of tissue damage could not be confirmed, and future studies should conduct histological analyses after exposure to suspended solids to compare these observations with the serum profile, further establishing a reference value.

In addition, this study did not assess pseudofeces generation, which represents a key physiological mechanism by which bivalves expel indigestible or non-nutritive particles. While pseudofeces production is known to play an important role in coping with suspended solids [42], our experimental design focused on biochemical markers (GOT, GPT, ALP, and cortisol) to evaluate internal stress responses rather than particle rejection behavior. As such, the present results mainly highlight the internal physiological consequences of suspended solid exposure. Future studies combining pseudofeces assessment with biochemical indicators would provide a more integrated understanding of how bivalves balance external particle rejection and internal stress regulation when subjected to suspended solids.

5. Conclusions

In this study, we conducted a physiological analysis of V. philippinarum and A. irradians in response to floaters. The physiological analytes examined in the experiment included GOT, GPT, ALP, and cortisol. The results revealed a significant upregulation of all these analytes after exposure to floaters. Additionally, we investigated mortality after floaters to assess the impact of another stimulus, water temperature stress, on their health. Mortality was observed only in the group exposed to floaters. Consequently, we posit that floaters have a detrimental effect on marine life.