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Article

Determination of Median Lethal Concentration (LC50) and Nitrite Accumulation in the Blood and Tissue of Blood Cockle (Tegillarca granosa, Linnaeus 1758)

by
Nurul Hazwani Hashim
1,
Ferdaus Mohamat-Yusuff
1,2,*,
Amirul Azuan Joni
1,
Faradiella Mohd Kusin
1,
Khairul Nizam Mohamed
1,
Zufarzaana Zulkeflee
1,
Zulfa Hanan Asha’ari
1 and
Syaizwan Zahmir Zulkifli
2,3
1
Department of Environment, Faculty of Forestry and Environment, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia
2
International Institute of Aquaculture & Aquatic Sciences (I-AQUAS), Universiti Putra Malaysia, Jalan Kemang 6, Batu 7, Teluk Kemang, Port Dickson 71050, Negeri Sembilan, Malaysia
3
Department of Biology, Faculty of Science, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia
*
Author to whom correspondence should be addressed.
Water 2020, 12(8), 2197; https://doi.org/10.3390/w12082197
Submission received: 20 May 2020 / Revised: 30 June 2020 / Accepted: 6 July 2020 / Published: 5 August 2020
(This article belongs to the Special Issue Impacts of Pollution on Coastal and Marine Ecosystems)
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Abstract

:
This study aimed to determine the nitrite toxicity of blood cockle Tegillarca granosa, with the objectives being to identify the median lethal concentration (LC50) and the accumulation level of nitrite in Tegillarca granosa, and to determine the relationship of nitrite accumulation with mortality percentage. The levels of LC50 and accumulation of nitrite were determined after 72 h of exposure to different nitrite concentrations (0, 0.5, 1.0, 1.5, and 2.0 mg/L). Nitrite accumulation was analysed using Method 8153 and a DR2800 spectrophotometer (HACH, Loveland, CO, USA). LC50 was identified at 1.53 mg/L, and nitrite accumulated in the ranges of 0.012 to 0.106 mg/L wet weight and 0.002 to 0.089 mg/L wet weight in the blood and soft tissue samples, respectively. Accumulation concentration in both tissue and blood cells increased proportionally with the exposure concentration, and had a strong positive relationship with the percentage of mortality. Our findings suggest that prolonged exposure of nitrite led to accumulation in the blood and tissues and caused cockle mortality.

1. Introduction

The presence of high nutrient residue in aquatic environments has been known to harm receiving water bodies. In addition, the presence of nutrient byproducts in water may cause aquatic life mortality. For example, the presence of nitrite (NO2−) due to the reduction of nitrates under conditions of an oxygen deficit increases the risk of accumulation of toxic nitrite, and may result in mass aquatic life mortality [1,2]. Examples of nitrite toxicity to aquatic lives are listed in Table 1. In a study of nitrite toxicity on post larvae of Penaeus setiferus (white shrimp), nitrite exposure significantly decreased the larvae’s temperature tolerance [3], while a study of amphibians showed nitrite-induced behavioural and morphological changes, reduced feeding activity, and also caused disequilibrium and paralysis, abnormalities, and death [4,5]. In fish, nitrite has resulted in the inhibition of chloride ion uptake at the bronchi, impairment of the acid–base balance and the electrolyte balance, reduction of the oxygen-carrying capacity of blood by the oxidation of haemoglobin (Hb) to methaemoglobin (MetHb), changes in gill histopathology and gross growth efficiency, and the survival of extracellular hyperkalaemia [6,7,8]. Moreover, the passage of nitrite into the bloodstream results in the irreversible conversion of Hb to MetHb, thus compromising its oxygen-binding capacity, causing respiratory deficiencies in aquatic animals and human beings [9,10].
Nitrite toxicity has been known to depend greatly on the salinity of the water, with less toxic effects evident in marine organisms compared to freshwater organisms. However, because estuaries and coastlines receive terrestrial effluent, and both water bodies are productive areas of benthic life, the presence of nitrite is expected to have an impact on benthic life. Due to poor understanding of the effects of nitrite in estuaries, therefore, there is a need to continuously monitor and study the effects of nitrites in marine and estuarine organisms. In addition, very limited information exists addressing the effects of nitrite on bivalves, particularly on blood cockles Tegillarca granosa (Linnaeus, 1758). The production of Tegillarca granosa has reportedly experienced a tremendous decline in Southeast Asian countries, particularly Malaysia, over the past decade [20]. Although the most common reason for the decline of cockle production is excessive nutrients from agricultural activities [21], limited studies have been conducted on the toxicity effect of nitrite on the species. Hence, this study was conducted to provide baseline information on the effect of nitrite on Tegillarca granosa by determining the median lethal concentration of nitrite and the level of nitrite accumulation in the blood and soft tissues of Tegillarca granosa, as well as to study the relationship between nitrite accumulations and cockle mortality.

2. Materials and Methods

2.1. Sampling

Samples of Tegillarca granosa were collected from Bagan Pasir Laut, which is located 10 km from Bagan Datoh, Perak, Malaysia (Figure 1). The coordinates for the sampling site are 3°51′20.556′′ N, 100°49′26.1444′′ E. Sampling was undertaken on 4 October 2018 to avoid the cockle’s spawning season. Samples of Tegillarca granosa were collected with a hand dredge net of 1.5 cm mesh. Approximately 100 cockles with sizes in the range 2–3 cm (Figure 1) were collected and transported alive to the laboratory.

2.2. Preparation of Artificial Seawater

Artificial seawater was used instead of natural seawater to minimize interference from other nutrients present in natural seawater that would affect the study findings. Artificial seawater with salinity between 25 to 30 ppt was prepared by dissolving 30 g of fine sea salt in 1 L of distilled water that was heated to 80 °C to accelerate the dissolving time, and then cooled to 28–35 °C. Other water parameters were monitored and controlled according to their natural habitat, as shown in Figure 2 and as in the Organization for Economic Co-operation and Development [22]. The oxygen content was monitored at a level of least 90% concentration (8.26–7.43 mg O2/L at 25 °C) after aeration and stabilization for 24 h [23].

2.3. Preparation of Nitrite Stock Solution

Stock solution of nitrite with concentration 100 mg/L was prepared by dissolving 0.15 g dried sodium nitrite (NaNO2) in artificial seawater and bringing to a volume of 1000 mL. Before preparing the stock solution, NaNO2 powder was dried at 105 °C and kept in a desiccator prior to utilization, in order to remove any excess moisture that would affect the result. The stock solution was prepared daily according to the Standard Method for the Examination of Water and Wastewater, Nitrogen (Nitrite) Section 4500-NO2 Colorimetric Method.

2.4. Cockle Acclimatization

The cockle samples were acclimatized for 24 h in 8 L of aerating artificial seawater, and the water was changed twice per day to simulate a suitable environment for cockles. The water quality parameters were monitored. Cockles were not fed, in order to excrete the contents of the digestive tract, including contaminants that may be associated with digested food and fecal matter, as well as to optimize the exposure rate [24]. This process is also known as depuration [24].

2.5. Cockle Exposure

A group of 45 blood cockles was selected randomly and used for the nitrite exposure test. Three individual cockles were placed in each treatment tank by 1 L beakers and exposed to 300 mL of the test solution. Each tank corresponded to different concentrations of nitrite (0, 0.5, 1.0, 1.5, and 2.0 mg/L), including a control group (0 mg/L) containing only artificial seawater. The exposure was undertaken for 72 h, and observations were recorded daily. The series of nitrite concentrations selected in the study were based on a 24 h range-finding test method. The test solutions for cockle exposure were replaced twice per day to maintain the test concentration and simulate the cockles’ natural environment. A static renewal water delivery system was applied throughout the exposure, and the exposure tanks were not aerated to minimise loss of nitrite content in the test solution, due to nitrite oxidation [25,26]. Cockles were not fed during the exposure in order to reduce nitrogen excretion, maintain water quality, and prevent other interference [27,28]. The death of a cockle was confirmed by its inability to close its valve upon mechanical stimulus, such as by touching with a glass rod, and it was then immediately removed from the treatment tank [29]. The essential water parameters were recorded and maintained: salinity 27 ± 2 ppt, dissolved oxygen above 7 mg/L, pH 7 ± 0.5, and temperature 30.5 ± 0.3 °C. The nitrite toxicity test was conducted in three replicates.

2.6. Nitrite Analysis

The blood and whole-body soft tissue samples were collected from cockles that survived the 72 h nitrite exposure. The blood samples were extracted from the forced opening of the cockles’ shells, and were obtained using a 1 cc insulin syringe fitted with a 29 G needle. Blood samples were pooled together according to the testing concentration in the ethylene diamine tetra-acetic acid (EDTA) bottles, centrifuged to obtain the plasma, and then 100 µL of the plasma used for ELISA reading. The tissue samples were pooled together according to the test concentration; homogenized in phosphate buffer saline (PBS), using a homogenizer; and centrifuged to obtain the supernatant. Then 100 µL of the supernatant was used for ELISA reading. If not processed immediately, both plasma and tissue supernatant samples were frozen at −80 °C until analysis to prevent nitrite oxidation. It is of note that all analyses (blood and soft tissue samples) were performed in three experimental replicates using an ELISA reader. This procedure was based on the Cayman Chemical Nitrate/Nitrite Colorimetric Assay Kit procedure manual.

2.7. Statistical Analysis

The 72 h LC50 was obtained by plotting the mortality percentage against the nitrite concentration using Probit analysis. The variation in nitrite concentrations of tested samples was performed using the t-test and the relationship of nitrite accumulation against exposure concentration, and the mortality percentage was conducted using linear regression analysis.

3. Results

Overall Results

The mortality percentage of Tegillarca granosa exposed to different concentrations of nitrite is shown in Figure 2. Findings indicate the mortality percentage proportionally increases with the concentration of nitrite exposure. The median lethal concentration (LC50) of nitrite in blood cockles was calculated to be 1.53 mg/L based on Probit analysis, and the 95% confident interval was between −1.76 and 4.56 mg/L, as shown in Table 2. The concentrations of nitrite in the blood and soft tissue samples ranged from 0.012 to 0.106 mg/L wet weight and 0.002 to 0.089 mg/L wet weight, respectively, as shown in Figure 3. No significant differences of nitrite concentration were found between the blood and soft tissue samples at each nitrite concentration, with p > 0.05. The relationship between nitrite accumulations in the cockles (blood and soft tissue) against the exposure concentration was performed using linear regression analysis, as shown in Table 3. A strong positive relationship was observed between the nitrite accumulation in the blood samples and the exposure concentration, with R2 = 0.8839 (Table 3). A point estimate for the true mean nitrite accumulation in the blood samples was 0.0458 mg/L wet weight, and the 95% confident interval was between 0.004 and 0.088 mg/L wet weight (Table 3). A strong positive relationship was observed between nitrite accumulation in the soft tissue and the exposure concentration, with R2 = 0.9876 (Table 3). The true mean nitrite accumulation in the soft tissue samples was 0.046 mg/L wet weight, and the 95% confident interval was between 0.033 and 0.06 mg/L wet weight (Table 3). The relationship between nitrite accumulation and cockle mortality was performed using linear regression analysis. A strong positive relationship was found between nitrite accumulation in the blood and the soft tissue, as well as the percentage of mortality, with R2 = 0.8482 and R2 = 0.9979, respectively (Figure 4).

4. Discussion

4.1. Median Lethal Concentration (LC50)

Due to limited information regarding the LC50 of nitrite in bivalves, the findings could not be compared with other results from the same species or the same phylum. In a study of the tremendous reduction of Tegillarca granosa production, the most common reason was given as excessive nutrients from agricultural activities [20]. Therefore, the LC50 of nitrite obtained in the present study proves the potential risk of nutrient contamination affecting bivalve communities within coastal areas.

4.2. Nitrite Accumulation in the Blood and the Soft Tissue

The lack of significant differences between nitrite concentration in the blood samples and the soft tissue samples (p > 0.05) at each exposure concentration is expected, due to the presence of blood cells in the wet soft tissue samples used in the analysis. Thus, the readings were almost equal between the two sets of the data (Figure 3). The soft tissue samples were not distinguished according to the organs, as this study represented the common human feeding habit in which the whole soft tissue of the cockle is consumed [30,31]. Findings were inconsistent with previous studies (of the same species), where a higher nitrite level was reported in the blood compared to the gills, liver, brain, and muscles [32,33]. This contradiction between the current findings and the literature is to be expected, because the concentrations in the cockle’s entire soft tissue would be higher than those in specific organs.
The strong positive relationship observed between the concentrations of nitrite in the cockle samples with the exposure concentration, as shown in Table 3, is expected, due to the possibility of nitrite ions diffusing into respiration organs and transferring to the whole body through the bloodstream [34,35]. Gills are known to act as an active site for the sorption of nutrients and pollutants [36] therefore, nitrite accumulation in the blood and soft tissue is directly proportional to the exposure concentration (Table 3). The possible risk due to the high accumulation upon entry is oxidization by nitrite of the iron in hemoglobin (Hb), resulting in methaemoglobinaemia in the cockles [28,37]. Methemoglobin (MetHb) under naked-eye observation is a dark brownish color when exposed to air, compared to the normal blood, which has a red color [38]. Dark-colored blood has undergone hemolysis—that is, the rupturing of red blood cells (RBCs) and release of their contents, causing RBCs to lose their properties and affecting their roles in the body system [28,29,30,31,32,33,34,35,36,37,38,39,40]. Methemoglobinemia prevents oxygen binding and transportation; hence, sample individuals with a high level of MetHb suffer functional anemia, owing to the reduction of the total oxygen-carrying capacity in the blood [28], thereby causing tissue hypoxia [41], in which a certain region of the tissue is deprived of adequate oxygen supply [42].
In this study, the nitrite-treated samples were found to have ruptured RBCs, as the blood sample turned a dark brownish color compared to the RBC of the control sample, which had an evenly distributed red color, suggesting that nitrite exposure groups experienced methemoglobinemia, as shown in Figure 5. Thus, this finding indicates that nitrite has the potential to cause methemoglobinemia, thereby reducing the oxygen-carrying capacity in blood and eventually leading to respiratory deficiency in blood cockles. In other words, the accumulation of nitrite in the tissue of blood cockle Tegillarca granosa leads to further consequences for the tissue, such as tissue damage [28].
A strong positive relationship between nitrite accumulation in the blood and tissue against the mortality percentage (Figure 4) suggests that the accumulation of nitrite in blood and tissue led to mortality of the cockles. The potential reason for the fatality is as discussed above. This is expected due to consistent exposure intensity and increased duration exposure; cockles eventually died due to methemoglobinemia and tissue hypoxia, thus leading to oxygen deficiency in the blood. It is important to know the LC50 value of nitrate in blood cockles, as this compound is expected to cause damage to red blood cells (RBCs) or hemolysis. Even at a low concentration with a prolonged exposure, this could become toxic and represent a hazard to cockles.
Based on the Interim National Water Quality Standard for Malaysia (INWQS), the allowable nitrite concentration in estuarine habitats (Class E) is 0.055 mg/L, and concentrations higher than this value will be toxic to aquatic lives [43]. Although the tested concentration used in this study was greater than the allowable concentration stated in the INWQS, the findings suggest that continuous monitoring of nitrite contamination is of great concern, due to the rapid development of anthropogenic activities leading to excessive nutrient pollution, particularly from the agriculture and aquaculture sectors in coastal areas.

5. Conclusions

This study identified that the LC50 of nitrite after 72 h of exposure in adult Tegillarca granosa was 1.53 mg/L. Furthermore, no significant differences were found for nitrite concentration in blood and wet soft tissue samples at each concentration exposure. It is revealed that there was a strong relationship between nitrate accumulation in blood and soft tissue and the mortality percentage, thus indicating that an accumulation of nitrite in the blood and soft tissue leads to the mortality of cockles. Hence, it can be said that these current findings serve as important baseline information for future studies. Areas with poor understanding and that require further investigation are the metabolism of nitrite in Tegillarca granosa and the effects of chronic exposure on the life stages of Tegillarca granosa.

Author Contributions

N.H.H. and F.M.-Y. were involved in planning, conceptualization, methodology, and supervision, and also performed the analysis and drafted the original manuscript. A.A.J. worked on reviewing and editing the manuscript; F.M.K., K.N.M., and Z.Z. were responsible for method validation and project administration; while Z.H.A. and S.Z.Z. aided in funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fundamental Research Grant Scheme 2017 (FRGS 2017), granted by the Malaysian Ministry of Higher Education, with the grant number 5540018.

Acknowledgments

The authors would like to thank laboratory assistants at Universiti Putra Malaysia for their sincere guidance and help with sampling. The cooperation from the Department of Fisheries Malaysia and representatives of cockles farmers, who cooperated throughout this project, is also not to be forgotten.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Svobodova, Z.; Kolarova, J. A review of the diseases and contaminant related mortalities of tench (Tinca tinca L.). Vet. Med. 2004, 49, 19–34. [Google Scholar] [CrossRef] [Green Version]
  2. Svobodova, Z.; Machova, J.; Poleszczuk, G.; Hůda, J.; Hamáčková, J.; Kroupova, H. Nitrite poisoning of fish in aquaculture facilities with water-recirculating systems. Acta Vet. Brno 2005, 74, 129–137. [Google Scholar] [CrossRef] [Green Version]
  3. Alcaraz, G.; Chiappa-Carrara, X.; Vanegas, C. Temperature tolerance of Penaeus setiferus postlarvae exposed to ammonia and nitrite. Aquat. Toxicol. 1997, 39, 345–353. [Google Scholar] [CrossRef]
  4. Marco, A.; Blaustein, A.R. The effects of nitrite on behavior and metamorphosis in Cascades frogs (Rana cascadae). Environ. Toxicol. Chem. 1999, 18, 946–949. [Google Scholar] [CrossRef] [Green Version]
  5. Marco, A.; Cash, D.; Belden, L.K.; Blaustein, A.R. Sensitivity to urea fertilization in three amphibian species. Arch. Environ. Contam. Toxicol. 2001, 40, 406–409. [Google Scholar] [CrossRef]
  6. Alcaraz, G.; Espina, S. Scope for growth of juvenile grass carp Ctenopharyngodon idella exposed to nitrite. Comp. Biochem. Physiol. Part C Pharmacol. Toxicol. Endocrinol. 1997, 116, 85–88. [Google Scholar] [CrossRef]
  7. Frances, J.; Allan, G.L.; Nowak, B.F. The effects of nitrite on the short-term growth of silver perch (Bidyanus bidyanus). Aquaculture 1998, 163, 63–72. [Google Scholar] [CrossRef]
  8. Grosell, M.; Jensen, F.B. Uptake and effects of nitrite in the marine teleost fish Platichthys flesus. Aquat. Toxicol. 2000, 50, 97–107. [Google Scholar] [CrossRef]
  9. Bradberry, S.M.; Gazzard, B.; Vale, J.A. Methemoglobinemia caused by the accidental contamination of drinking water with sodium nitrite. J. Toxicol. Clin. Toxicol. 1994, 32, 173–178. [Google Scholar] [CrossRef]
  10. Van Leeuwen, F.X.R. Safe drinking water: The toxicologist’s approach. Food Chem. Toxicol. 2000, 38, S51–S58. [Google Scholar] [CrossRef]
  11. Parra, G.; Yúfera, M. Tolerance response to ammonia and nitrite exposure in larvae of two marine fish species (gilthead seabream Sparus aurata L. and Senegal sole Solea senegalensis Kaup). Aquac. Res. 1999, 30, 857–863. [Google Scholar] [CrossRef]
  12. Meade, M.E.; Watts, S.A. Weight gain and survival of juvenile Australian crayfish Cherax quadricarinatus fed formulated feeds. J. World Aquac. Soc. 1995, 26, 469–474. [Google Scholar] [CrossRef]
  13. Kamstra, A.; Span, J.A.; Weerd, J.V. The acute toxicity and sublethal effects of nitrite on growth and feed utilization of European eel, Anguilla anguilla (L.). Aquac. Res. 1996, 27, 903–911. [Google Scholar] [CrossRef]
  14. Fontenot, Q.C.; Isely, J.J.; Tomasso, J.R. Uptake, inhibition, and depuration of nitrite to shortnose sturgeon Acipenser brevirostrum fingerlings. In Fish Response to Toxic Environments, Proceedings of the International Congress on the Biology of Fish, Towson University, Baltimore, MD, USA, 26–30 July 1998; American Fisheries Society: Vancouver, BC, Canada, 1998; p. 127. [Google Scholar]
  15. Atwood, H.L.; Fontenot, Q.C.; Tomasso, J.R.; Isely, J.J. Toxicity of nitrite to Nile tilapia: Effect of fish size and environmental chloride. N. Am. J. Aquac. 2001, 63, 49–51. [Google Scholar] [CrossRef]
  16. Marco, A.; Quilchano, C.; Blaustein, A.R. Sensitivity to nitrate and nitrite in pond-breeding amphibians from the Pacific Northwest, USA. Environ. Toxicol. Chem. Int. J. 1999, 18, 2836–2839. [Google Scholar] [CrossRef]
  17. Chen, J.C.; Chin, T.S. Acute toxicity of nitrite to tiger prawn, Penaeus monodon, larvae. Aquaculture 1988, 69, 253–262. [Google Scholar] [CrossRef]
  18. Alcaraz, G.; Chiappa-Carrara, X.; Espinoza, V.; Vanegas, C. Acute toxicity of ammonia and nitrite to white shrimp Penaeus setiferus postlarvae. Aquat. Toxicol. 1997, 39, 345–353. [Google Scholar] [CrossRef]
  19. Huo, Z.; Rbbani, M.G.; Tian, Y.; Wang, Y.; Yang, F.; Fang, L.; Yan, X. Nitrite tolerance of larvae and juveniles of geoduck clam Panopea japonica. Aquac. Res. 2018, 49, 2913–2917. [Google Scholar] [CrossRef]
  20. Department of Fisheries Malaysia (DOFM). Available online: https://www.dof.gov.my/ (accessed on 26 April 2018).
  21. Yurimoto, T.; Faizul, M.K.; Fuseya, R.; Alias, M. Mass mortality event of the blood cockle, Anadara granosa, in aquaculture ground along Selangor coast, Peninsular Malaysia. Int. Aquat. Res. 2014, 6, 177–186. [Google Scholar] [CrossRef] [Green Version]
  22. Organization for Economic Co-operation and Development Guidance (OECD). Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment. In OECD Environmental Health and Safety Publications; Series on Testing and Assessment No. 34; Organisation for Economic Co-operation and Development: Paris, France, 2005. [Google Scholar]
  23. Abu Shaala, N.M.; Zulkifli, S.Z.; Ismail, A.; Azmai, M.N.A.; Mohamat-Yusuff, F. Lethal Concentration 50 (LC50) and Effects of Diuron on Morphology of Brine Shrimp Artemia salina (Branchiopoda: Anostraca) Nauplii. Procedia Environ. Sci. 2015, 30, 279–284. [Google Scholar] [CrossRef] [Green Version]
  24. McLeod, C.; Polo, D.; Saux, J.C.L.; Guyader, F.S.L. Final Report: Evaluating the Effectiveness of Depuration in Removing Norovirus from Oysters; Seafood Safety Assessment Ltd.: Swindon, UK, 2017; pp. 15–16. [Google Scholar]
  25. Wetzel, R.G. Limnology of Lake and River Ecosystem, 3rd ed.; Academic Press: San Diego, CA, USA, 2001. [Google Scholar]
  26. Simmons, A.E.; Karimi, I.; Talwar, M.; Simmons, T.W. Effects of nitrite on development of embryos and early larval stages of zebrafish (Danio rerio). Zebrafish 2012, 9, 200–206. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Silberberg, M. Chemistry: The Molecular Nature of Matter and Change; McGraw-Hill Higher Education: New York, NY, USA, 2004. [Google Scholar]
  28. Huertas, M.; Gisbert, E.; Rodrıguez, A.; Cardona, L.; Williot, P.; Castello-Orvay, F. Acute exposure of Siberian sturgeon (Acipenser baeri, Brandt) yearlings to nitrite: Median-lethal concentration (LC50) determination, haematological changes and nitrite accumulation in selected tissues. Aquat. Toxicol. 2002, 57, 257–266. [Google Scholar] [CrossRef]
  29. Reddy, N.A.; Menon, N.R. Effects of ammonia and ammonium on tolerance and byssogenesis in Perna viridis. Mar. Ecol. Prog. Ser. 1979, 1, 315–321. [Google Scholar] [CrossRef]
  30. Figueira, E.; Lima, A.; Branco, D.; Quintino, V.; Rodrigues, A.M.; Freitas, R. Health concerns of consuming cockles (Cerastoderma edule L.) from a low contaminated coastal system. Environ. Int. 2011, 37, 965–972. [Google Scholar] [CrossRef] [PubMed]
  31. Rainbow, P.S. Trace Metals in the Environment and Living Organisms: The British Isles as a Case Study; Cambridge University Press: Cambridge, UK, 2018. [Google Scholar]
  32. Bodansky, O. Methemoglobinemia and methemoglobin-producing compounds. Pharmacol. Rev. 1951, 3, 144–196. [Google Scholar]
  33. Margiocco, C.; Arillo, A.; Mensi, P.; Shenone, G. Nitrite bioaccumulation in Salmo gairdneri and haematological consequences. Aquat. Toxicol. 1983, 3, 261–270. [Google Scholar] [CrossRef]
  34. Vahl, O. Porosity of the gill, oxygen consumption and pumping rate in (Cardium edule L.)(Bivalvia). Ophelia 1972, 10, 109–118. [Google Scholar] [CrossRef]
  35. Ibarrola, I.; Larretxea, X.; Navarro, E.; Iglesias, J.I.P.; Urrutia, M.B. Effects of body-size and season on digestive organ size and the energy balance of cockles fed with a constant diet of phytoplankton. J. Comp. Physiol. B 2008, 178, 501–514. [Google Scholar] [CrossRef]
  36. Eapen, J.T.; Patel, B. Haematological evaluation of naphthalene intoxication in the tropical arcid blood clam Anadara granosa. Mar. Biol. 1989, 100, 223–226. [Google Scholar] [CrossRef]
  37. Stoppler, M.C.; Davis, C.P. Acquired Methemoglobinemia. MedicineNet. Available online: https://www.medicinenet.com/acquired_methemoglobinemia/views.html (accessed on 26 April 2018).
  38. Malkarnekar, S.B.; Anjanappa, R.; Naveen, L.; Kiran, B.G. Acute methemoglobinemia with haemolytic anaemia following bio-organic plant nutrient compound exposure: Two case reports. Indian J. Crit. Care Med. 2014, 18, 115–117. [Google Scholar] [CrossRef]
  39. Campbell, T.W.; Ellis, C.K. Avian and Exotic Animal Haematology and Cytology, 3rd ed.; Blackwell Publishing: Hoboken, NJ, USA, 2007; pp. 3–34. [Google Scholar]
  40. The Joint Pathology Center. Urinary System; The Joint Pathology Center: Silver Spring, MD, USA, 2015; Available online: https://www.askjpc.org/vspo/show_page.php?id=VFN3WmJBZVVxeFFkempVMll2YzFOUT09 (accessed on 20 September 2018).
  41. Noga, E.J. Fish Disease: Diagnosis and Treatment, 2nd ed.; Wiley: Hoboken, NJ, USA, 2010. [Google Scholar]
  42. Fago, A.; Jensen, F.B. Hypoxia tolerance, nitric oxide and nitrite: Lessons from extreme animals. Physiology 2015, 30, 116–126. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Svobodova, Z.; Lloyd, R.; Machova, J.; Vykusova, B. Water Quality and Fish Health; EIFAC Technical Paper, No. 54; Food and Agriculture Organization: Rome, Italy, 1993; pp. 16–19. [Google Scholar]
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Figure 1. Tegillarca granosa samples used for nitrite exposure.
Figure 1. Tegillarca granosa samples used for nitrite exposure.
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Figure 2. Mortality (%) of Tegillarca granosa after 72 h of exposure to a series of nitrite (NO2-N) concentrations.
Figure 2. Mortality (%) of Tegillarca granosa after 72 h of exposure to a series of nitrite (NO2-N) concentrations.
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Figure 3. Mean concentration of nitrite in the blood and tissue of exposed blood cockles, with p > 0.05.
Figure 3. Mean concentration of nitrite in the blood and tissue of exposed blood cockles, with p > 0.05.
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Figure 4. Blood cockle mortality percentages vs. the concentration of nitrite in the blood (a) and soft tissue (b) samples of nitrite-treated cockles.
Figure 4. Blood cockle mortality percentages vs. the concentration of nitrite in the blood (a) and soft tissue (b) samples of nitrite-treated cockles.
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Figure 5. Differences of the extracted blood samples’ color between the nitrite exposed sample and the control group.
Figure 5. Differences of the extracted blood samples’ color between the nitrite exposed sample and the control group.
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Table
Table 1. Examples of the nitrite toxicity to aquatic fauna.
Table 1. Examples of the nitrite toxicity to aquatic fauna.
Species TestNitrite (mg/L)Reference
Gilthead seabream (12-day-old larvae)LC50: 24 h607[11]
Australian crayfishLC50: 24 h42.9[12]
European eelLC50: 96 h144[13]
Juvenile grass carpLC50: 96 h10.6[6]
Short-nose sturgeonLC50: 96 h10[14]
Nile tilapiaLC50: 96 h8–81[15]
AmphibianLC50: 15 d<2[16]
Tiger prawnLC50: 96 h14[17]
White shrimp (Penaeus setiferus) postlarvaeLC50: 24 h
LC50: 48 h
LC50: 72 h		1.49
1.21
1.12		[18]
Geoduck clam
(Panopea japonica) juvenile		LC50: 96 h112.76[19]
Table
Table 2. Estimated concentration–response curves of Tegillarca granosa mortality after 72 h of exposure to nitrite a.
Table 2. Estimated concentration–response curves of Tegillarca granosa mortality after 72 h of exposure to nitrite a.
InterceptStandard ErrorSlopeLC5095% Confidence Interval
1.400.992.361.53(−1.76, 4.56)
a the median lethal concentration (LC50) and the corresponding 95% confidence limits are reported.
Table
Table 3. Estimated nitrite accumulation curves of Tegillarca granosa after 72 h of exposure to nitrite b.
Table 3. Estimated nitrite accumulation curves of Tegillarca granosa after 72 h of exposure to nitrite b.
SampleMean (mg/L) Wet WeightInterceptStandard ErrorSlopeR295% Confidence Interval
Blood0.0458−0.00020.0150.0460.8839(0.004, 0.088)
Soft Tissue0.0460.00020.0050.04580.9876(0.033, 0.06)
b the corresponding 95% confidence limits are reported.

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Hashim, N.H.; Mohamat-Yusuff, F.; Joni, A.A.; Mohd Kusin, F.; Mohamed, K.N.; Zulkeflee, Z.; Asha’ari, Z.H.; Zahmir Zulkifli, S. Determination of Median Lethal Concentration (LC50) and Nitrite Accumulation in the Blood and Tissue of Blood Cockle (Tegillarca granosa, Linnaeus 1758). Water 2020, 12, 2197. https://doi.org/10.3390/w12082197

AMA Style

Hashim NH, Mohamat-Yusuff F, Joni AA, Mohd Kusin F, Mohamed KN, Zulkeflee Z, Asha’ari ZH, Zahmir Zulkifli S. Determination of Median Lethal Concentration (LC50) and Nitrite Accumulation in the Blood and Tissue of Blood Cockle (Tegillarca granosa, Linnaeus 1758). Water. 2020; 12(8):2197. https://doi.org/10.3390/w12082197

Chicago/Turabian Style

Hashim, Nurul Hazwani, Ferdaus Mohamat-Yusuff, Amirul Azuan Joni, Faradiella Mohd Kusin, Khairul Nizam Mohamed, Zufarzaana Zulkeflee, Zulfa Hanan Asha’ari, and Syaizwan Zahmir Zulkifli. 2020. "Determination of Median Lethal Concentration (LC50) and Nitrite Accumulation in the Blood and Tissue of Blood Cockle (Tegillarca granosa, Linnaeus 1758)" Water 12, no. 8: 2197. https://doi.org/10.3390/w12082197

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