Toxicity of Nitrite to Juvenile Sunray Surf Clam (Mactra chinensis Philippi)

Abstract

Nitrite is a common pollutant in marine environments and can cause mortality in crustaceans and bivalves. The purpose of the current study is to understand nitrate’s toxicity to juvenile clams due to its potential impact on aquaculture and marine ecosystems. Juvenile sunray surf clams (Mactra chinensis Philippi) (1.00 ± 0.10 cm shell length, 0.75 ± 0.04 cm shell height) were exposed to varying concentrations of nitrite for 96 h and 20 days, respectively. The LC₅₀ for survival at 96 h was 37 mg/L NO₂-N. Histological evaluations were made on juvenile clams exposed at 30 mg/L after 20 d of exposure. Epithelial cells and digestive diverticulum are the best sub-lethal effect indicators. Shell length and antioxidant enzyme activities were measured at the beginning of the experiment and then observed 10 and 20 days after exposure. A logarithmic relationship was obtained between the relative growth rate (based on the shell length) of juvenile M. chinensis and the nitrite concentration. Compared to the control, activity suppression of superoxide dismutase and catalase activity was detected from the concentration of 1 mg/L NO₂-N. It is recommended that nitrite concentrations remain below 1 mg/L to prevent stress during the early developmental stages of clams.

1. Introduction

Clams are sensitive indicators of sediment and water toxicity [1], and clam aquaculture could be considered a net carbon sink [2]. The sunray surf clam, also known as the Hen Clam, is a commercially essential bivalve species that has experienced population declines due to overfishing and coastal habitat degradation [3]. It is a rich source of protein and minerals, offering high nutritive value and serving as an important food source in Southeast Asia [4]. It is a dioecious species with a spawning period from June to August, and its reproductive behavior and embryonic development have been studied [5].

Nitrite (NO₂⁻) is a critical component of the nitrification process in marine environments [6]. While typically present in low concentrations, elevated levels can occur as a result of effluents or disrupted nitrification processes, leading to health deterioration in aquatic organisms [7]. Studies have demonstrated that elevated nitrite concentrations can cause acute and chronic toxic effects in aquatic species, such as white shrimp [8]. In aquaculture, nitrite exposure can result in gill lesions and impair oxygen transport in the blood, leading to respiratory issues in fish [9]. Sensitivity to nitrite varies across species, with some being more susceptible than others [10]. Given the potential for nitrite to disrupt physiological functions and harm clams, further research is needed to fully understand its impact on these organisms.

Bivalves are sensitive to pollutants and are considered “ocean sentinels” [11]. A few studies have investigated nitrite’s toxic effects on bivalves. For instance, research has shown that nitrate–nitrogen pollution can negatively affect the population status of freshwater mussels [12]. However, the toxicity of nitrite to marine species, particularly clams, remains understudied compared to that in freshwater species [13]. Widman et al. exposed juvenile bay scallops (Argopecten irradians irradians) for 72 h and observed 100% mortality at nitrite concentrations of 800 mg N/L [14]. Lv reported that nitrite exposure can cause oxidative damage, suppressed immune function, and impaired tissue regeneration in clams (Ruditapes philippinarum) [15].

The toxicity of nitrite can vary depending on the species and environmental conditions [16]. Further research is needed to clarify the specific effects of nitrite on marine organisms and to explore nitrite resistance in species that are being considered for aquaculture [17]. This study aimed to determine nitrite’s lethal and sub-lethal toxicity in sunray surf clam (Mactra chinensis Philippi). Recommendations on suitable nitrite concentrations range from aquaculture systems are suggested based on determined toxicity levels.

2. Materials and Methods

2.1. Test Organisms and Exposure Conditions

Juvenile clams (Mactra chinensis Philippi) were bred from parent clams sourced from a marine clam farming site in Zhuanghe (Dalian, Liaoning, China). These parent clams were originally collected from the natural sea area of Jianshan (Dalian, Liaoning, China). Upon transport to the laboratory, healthy clams with uniform shell length (SL, 1.00 ± 0.10 cm) and shell height (SH, 0.75 ± 0.04 cm) were selected for the experiment after a minimum two-week acclimation period. Healthy clams are characterized by intact, smooth shells that are free from pathogens or parasites. Additional indicators include bright, clear soft tissue with no discoloration and a strong shell closure reflex when disturbed. Seawater was sourced from the Heishijiao mariculture area (38°87′ N, 121°55′ E, Dalian, Liaoning, China). During the nitrite toxicity test, the seawater pH (8.18 ± 0.07) and temperature (19.3 ± 0.6 °C) were maintained at constant levels. Dissolved oxygen (DO) and ammonia nitrogen (NH⁺₄-N) were monitored regularly. The DO and NH⁺₄-N in the solution ranged between 6.0 and 8.0 mg/L and 0.269 to 0.468 mg/L, respectively. The photoperiod was 16 h of light and 8 h of dark.

The stock solution was prepared daily by dissolving the required amount of sodium nitrite (NaNO₂, 20 mg/mL, analytical reagent, Shenyang Chemical Reagent Co., Shenyang, China) in test water. Nitrite concentrations were measured according to the National Standards of the People’s Republic of China for marine monitoring (GB 17378.1-2007 [18]) using a UV–visible spectrophotometer (V-1800, Shanghai Mapade Instruments, Shanghai, China). To prevent nitrite oxidation, no aeration was provided during the exposure periods. Three replicates, each containing 20 juvenile clams, were tested in separate 2 L glass beakers and observed daily for mortality. Dead clams were removed at 24 h intervals, and the exposure water was changed daily. Additional information on test organisms and water quality can be found in previous reports [19,20].

2.2. Experimental Design

The nine nominal nitrite levels for acute exposure (96 h) were 0.6, 10, 20, 40, 70, 100, 200, 500, and 1000 mg/L NO₂-N, along with a control. The pH, water temperature, and salinity were monitored daily. Nitrite levels were measured at the start and after 24, 48, and 96 h in all concentrations, as nitrite can easily be converted to nitrate in the presence of sufficient dissolved oxygen. Juvenile clams were not fed during the acute bioassays.

For sub-lethal exposure (20 d), six nominal nitrite concentrations (1, 2, 4, 8, 15, 30 mg/L NO₂-N) and a control were tested in triplicate to assess the effects of nitrite on survival and growth in juvenile clams. The clams were fed with a daily algae mixture (1% v/v). Survival and growth were evaluated after 96 h, and again at 10 and 20 days. Enzyme activity for superoxide dismutase (SOD) and catalase (CAT) was measured according to the manufacturer’s instructions (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China) using homogenized soft tissue from surviving clams. For histological analysis, we selected live clams that had been exposed to 30 mg/L nitrite for 20 days. Additional experimental details are available in previous reports [19,20].

2.3. Data Analysis

The mean actual nitrile concentrations for each bioassay were used to calculate LC values. All statistical analyses were performed using SPSS Version 13.0, and data are presented as means ± standard deviation (SD). Differences between treatments were analyzed using one-way analysis of variance (ANOVA) with the least significant difference (LSD) method for multiple comparisons, followed by Duncan’s multiple range test.

Statistical significance was set at p < 0.05. The LC₅₀ values, along with a 95% credible interval (CI), were estimated using a freely available internet platform, MOSAIC [21]. The relative growth rate (RGR) was calculated using the following equation:

$$\mathrm{R}\mathrm{G}\mathrm{R}={\displaystyle \frac{\mathrm{L}1-\mathrm{L}0}{\mathrm{L}0}}$$

(1)

where L0 and L1 are the shell lengths measured before and after exposure, respectively. The safe concentration (SC) of nitrite for juvenile M. chinensis was estimated by applying an application factor of 0.1 to the 96 h LC₅₀ value [22].

3. Results

3.1. Lethal Toxicity Test

The preset and measured nitrite concentrations are presented in

Table 1. Nitrite concentrations (mean ± SD, n = 3, mg/L) during acute exposure.

Exposure Period	Renewal	Control	1	2	3	4	5	6	7	8	9
Preset		0	0.6	10	20	40	70	100	200	500	1000
0 h		0.39	0.63	9.90	19.23	38.22	68.22	97.00	196.87	499.67	998.76
24 h	Before	0.41 ± 0.01	0.44 ± 0.08	8.94 ± 0.33	17.69 ± 0.33	32.66 ± 0.88	62.94 ± 2.20	94.60 ± 2.20	177.69 ± 4.40	453.62 ± 30.46	826.07 ± 51.89
	After	0.58	0.56	9.61	19.80	39.37	66.78	98.44	199.75	499.67	N/A
48 h	Before	0.39 ± 0.00	0.49 ± 0.00	8.56 ± 0.17	17.21 ± 0.29	31.12 ± 0.66	61.98 ± 0.83	91.72 ± 1.66	171.93 ± 3.32	480.48 ± 6.65	N/A
	After	0.57	0.63	9.90	19.23	39.95	69.65	99.88	199.75	N/A	N/A
96 h		0.40 ± 0.01	0.44 ± 0.08	8.85 ± 0.33	18.46 ± 1.45	35.54 ± 4.16	64.86 ± 3.00	97.48 ± 0.83	192.08 ± 1.66	N/A	N/A

Note: N/A indicates that levels were not measured due to the mortality of juvenile clams. Concentrations of the bulk solution were measured after each water renewal.

. Desired nitrite levels reached the nominal concentrations at the start of the experiment and prior to each water renewal. The temperature, salinity and pH were maintained at 19.85 ± 0.53 °C, 30 psu, and 8.17–8.19, respectively.

Mortality rates at different nitrite concentrations after 96 h are shown in Figure 1. Increasing nitrite levels had a detrimental effect on clam survival, with mortality reaching 100% at a nominal concentration of 1 000 mg/L at 24 h and 500 mg/L at 48 h, respectively. The LC₅₀ values at 24, 48, and 96 h were 133.76 (93.52, 191.30) mg/L, 63.40 (47.33, 84.92) mg/L, and 36.93 (27.54, 49.52) mg/L, respectively.

3.2. Sub-Lethal Toxicity Test

Nitrite concentrations at each sampling point for each treatment are summarized in

Table 2. Nitrite concentrations (mean ± SD, n = 6, three samples taken before and after water renewal, mg/L) during sub-lethal experiments.

Nominal Concentration	Measured Concentration (mg/L)
	24 h	48 h	96 h	10 d	20 d
Control	0.47 ± 0.01	0.47 ± 0.00	0.50 ± 0.00	0.52 ± 0.00	0.54 ± 0.01
1.0 mg/L	0.83 ± 0.10	0.92 ± 0.04	1.05 ± 0.04	1.05 ± 0.04	0.90 ± 0.04
2.0 mg/L	1.79 ± 0.08	2.10 ± 0.08	1.84 ± 0.08	2.06 ± 0.15	1.84 ± 0.08
4.0 mg/L	3.73 ± 0.12	3.96 ± 0.08	4.13 ± 0.02	4.05 ± 0.08	3.96 ± 0.08
8.0 mg/L	7.96 ± 0.03	8.01 ± 0.26	7.95 ± 0.06	7.83 ± 0.15	8.06 ± 0.05
15.0 mg/L	14.64 ± 0.26	15.08 ± 0.15	14.55 ± 0.15	14.90 ± 0.00	15.20 ± 0.07
30.0 mg/L	30.19 ± 0.15	29.57 ± 0.15	30.19 ± 0.15	29.63 ± 0.13	29.67 ± 0.03

. The target nitrite levels were achieved within acceptable deviations across treatments. For consistency, subsequent descriptions of both lethal and sub-lethal tests use nominal descriptions. No apparent mortality was observed at the highest concentrations, with survival rates ranging from 96.7% to 100%. After 20 days, survival rates across all nitrite concentrations were between 95% and 100%. Temperature and pH were maintained between 19.0 and 19.6 °C and 8.16–8.20, respectively.

The RGR decreased as the nitrite concentration increased (Figure 2). A logarithmic relationship was observed between the RGR of juvenile M. chinensis and nitrite concentration, with R² = 0.999 for 10 days and R² = 0.962 for 20 days, respectively. In the control group, the juvenile clams grew to 1.50 ± 0.03 cm and 1.89 ± 0.01 cm in SL on days 10 and 20, respectively. In contrast, at 30 mg/L nitrite, the mean SL was 1.37 ± 0.01 cm and 1.70 ± 0.01 cm, respectively. On day 20, juvenile clams exposed to 30 mg/L nitrite had a lower SH of 0.99 ± 0.02 cm compared to 1.22 ± 0.02 cm in the control group.

3.3. Antioxidant Enzyme Activities under Sub-Lethal Exposure

Figure 3 and Figure 4 show the activities of antioxidant enzymes (CAT and SOD) measured in the soft tissue of the juvenile clams. Both enzyme activities decreased with increasing nitrite concentrations. The activities of CAT and SOD were significantly lower in the juvenile clams exposed to ≥1 mg/L nitrite than in the control. At 30 mg/L nitrite on day 20, the mean activities of CAT and SOD were 33% and 41% of those observed in the Control, respectively. There was no increase in the activity of either of the enzymes after any of the treatments.

3.4. Histological Observations

Figure 5 illustrates the changes in the soft tissue structure of juvenile clams after 20 days of nitrite exposure. In healthy juvenile clams (control), the epithelial cells of the foot were tightly and regularly arranged, with well-structured muscle fibers and normal connective tissue morphology (Figure 5-1). However, at 30 mg/L nitrite concentration, epithelial cells showed signs of rupture and shedding (Figure 5-2). In the control group, the digestive diverticulum had a distinct structure, with neatly arranged acini interspersed with connective tissue (Figure 5-3). However, the digestive diverticulum contained vacuolated acini under the highest amount of nitrile exposure (Figure 5-4). In the control, the epithelial cells, muscle fibers, and connective tissue structures in the mantle are visible, with cells arranged in an orderly manner (Figure 5-5). However, the mantle epithelium in nitrite-impaired juvenile clams produces many basophilic cells that cluster together, causing other tissue structures to become blurred and disorganized (Figure 5-6).

4. Discussions

Juvenile M. chinensis shows lower tolerance to nitrite compared to some other mollusks. The 96 h lethal tolerance limits for hard clam (Mercenaria mercenaria) and the oyster (Crassostrea virginica, both juvenile and adult) ranged from 1081 to 2415 mg/L [23]. In comparison, the 24, 48, and 96 h LC₅₀ concentrations for the New Zealand Mud Snail (Potamopyrgus antipodarum) were reported as 2134, 848, and 535 mg/L NO₂-N, respectively [24], which is one order of magnitude higher than the current study (96 h LC₅₀ = 36.93 mg/L). Typically, the 96 h LC₅₀ values for nitrite–nitrogen in marine invertebrates range from 10 to 300 mg/L [9], consistent with the findings of this study.

Although limited information is available on nitrite guidelines created to protect marine life, it is known that nitrite toxicity is influenced by factors such as pH, salinity (chloride), and species sensitivity [25]. For marine species, safe nitrite-N levels have been reported as 0.71 mg/L (33 psu, pH = 8.20) for shrimp larvae [26] and 3.8 mg/L (20 psu, pH = 7.70) for juvenile shrimp [27]. Based on 10% of the 96 h LC₅₀, the safe concentration derived from the current study is 3.7 mg/L (30 psu, pH = 8.18), which aligns with the reported safe concentration range of 0.5 to 15.5 mg for marine invertebrates [9].

DO levels could also potentially affect the toxicity of nitrite. To the best of the authors’ knowledge, specific DO requirements for M. chinensis have not been studied directly. However, studies on related species suggest normal larval growth occurs at DO levels ≥4.2 mg/L for the hard clam (Mercenaria mercenaria) [28]. This indicates that an adequate DO level (6–8 mg/L) was maintained in the current study. Ammonia is another factor which could potentially contribute to toxicity interference in sensitive toxicity tests as a confounding factor [29]. However, the highest NH⁺₄-N level observed is lower than 0.5 mg/L, lower than the threshold of the protection of M. chinensis from ammonia (0.8 mg/L) in the literature [20].

Chronic or sub-lethal exposure to nitrite can inhibit growth and induce various physiological effects in marine invertebrates. For example, chronic exposure to nitrite levels above 6.67 mg/L NO₂-N has been shown to negatively affect shrimp growth [30]. Similarly, shrimp exposed to 4 mg/L nitrite for 2 days experienced reduced growth, though survival was unaffected [8], mirroring the findings of the current study. In general, nitrate concentrations and specific growth rates exhibit an exponential relationship [7], as observed in this study.

Variations in sensitivity during lethal and sub-lethal tests were observed in the current study, which could plausibly be due to genetic differences between individual clams of the same species [31]. Since juvenile clams were bred at constant environmental conditions for generations, variations in sensitivity were unlikely to have been caused by seasonal variations in the conditions of clams [32]. In future studies, researchers should consider including a positive control with a reference toxicant to improve the precision of the toxicity results obtained and further evaluate the sensitivity of M. chinensis in comparison to other bivalves [33]. Copper could be a potential candidate, as several studies in the literature addressed the sensitivity of mollusks to copper [34]. For example, CuSO₄ was used to evaluate the sensitivity of bivalve Gaimardia trapesina and gastropod Laevilittorina caliginosa [35].

Nitrite exposure in marine clams induces oxidative stress, leading to increased production of reactive oxygen species (ROS) and alterations in antioxidant enzyme activities. For instance, antioxidant enzyme activity in the intestines of Pacific white shrimp (Litopenaeus vannamei) decreased after 72 h of nitrite stress [36]. Similarly, nitrite exposure suppressed SOD and CAT activities in mud crabs (Scylla paramamosain) after 48 and 72 h of exposure [37]. While an initial increase in antioxidant enzyme activity has been observed in fish exposed to acute nitrite concentrations [38], the increase was not seen in the present study. This discrepancy could be due to the prolonged exposure in the current sub-lethal condition, as the initial enzyme increase may occur when the antioxidant system is insufficiently effective to neutralize excessive ROS [38]. Nitrite-induced oxidative stress has been reported in red claw crayfish (Cherax quadricarinatus) in a time-dependent manner [39]. The glutathione system, which plays a crucial role in the oxidative stress response to nitrite exposure [40], will be the focus of future research.

Histological observations in marine clams exposed to nitrite and other pollutants reveal various tissue alterations. For example, histological damage to the digestive diverticula has been reported in mussels exposed to N-nitrosodimethylamine [41]. Digestive gland lesions are considered one of the most reliable indicators of sub-lethal effects in the Baltic Clam (Macoma balthica) exposed to sediments contaminated by polynuclear aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and metals [42]. Although reproduction was not investigated in the current study, the loss of epithelial cells could lead to tissue dysfunction, potentially compromising reproductive capabilities over a short period [43].

5. Conclusions

This study investigated the effects of nitrite exposure on the survival, growth, and antioxidant enzyme activities of juvenile clams. Our results indicate that the LC₅₀ ranged from 37 to 134 mg/L NO₂-N over 24 to 96 h. While nitrite concentrations up to 30 mg/L may not be immediately lethal to juvenile clams (Mactra chinensis Philippi), exposure to as little as 1 mg/L NO₂-N for 10 days led to alterations in the clams’ antioxidant defense systems. Based on these findings, we recommend that nitrite concentrations not exceed 1 mg/L to prevent stress during the early development of clams. This study offers insights into the toxicological mechanisms of nitrite in marine organisms and highlights its potential impacts on aquaculture and ecosystem health.