water Article Determination of Median Lethal Concentration (LC50) and Nitrite Accumulation in the Blood and Tissue of Blood Cockle (Tegillarca granosa, Linnaeus 1758) 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; [email protected] (N.H.H.); [email protected] (A.A.J.); [email protected] (F.M.K.); [email protected] (K.N.M.); [email protected] (Z.Z.); [email protected] (Z.H.A.) 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; [email protected] 3 Department of Biology, Faculty of Science, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia * Correspondence: [email protected]; Tel.: +60-13-377-5514 Received: 20 May 2020; Accepted: 6 July 2020; Published: 5 August 2020 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. Keywords: blood cockle; bivalves; bio-accumulator; nitrite; toxicity; pollution 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 Table1. 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 Water 2020, 12, 2197; doi:10.3390/w12082197 www.mdpi.com/journal/water Water 2020, 12, 2197 2 of 10 (MetHb), changes in gill histopathology and gross growth efficiency, and the survival of extracellular hyperkalaemia [6–8]. 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]. Table 1. Examples of the nitrite toxicity to aquatic fauna. Species Test Nitrite (mg/L) Reference Gilthead seabream (12-day-old larvae) LC50: 24 h 607 [11] Australian crayfish LC50: 24 h 42.9 [12] European eel LC50: 96 h 144 [13] Juvenile grass carp LC50: 96 h 10.6 [6] Short-nose sturgeon LC50: 96 h 10 [14] Nile tilapia LC50: 96 h 8–81 [15] Amphibian LC50: 15 d <2 [16] Tiger prawn LC50: 96 h 14 [17] LC50: 24 h 1.49 White shrimp (Penaeus setiferus) postlarvae LC50: 48 h 1.21 [18] LC50: 72 h 1.12 Geoduck clam LC50: 96 h 112.76 [19] (Panopea japonica) juvenile 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 (Figure1). The coordinates for the sampling site are 3 ◦51020.55600 N, 100◦49026.144400 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 (Figure1) were collected and transported alive to the laboratory. Water 2020, 12, x FOR PEER REVIEW 3 of 10 Water 20202020,, 1212,, 2197x FOR PEER REVIEW 3 of 10 Figure 1. Tegillarca granosa samples used for nitrite exposure. Figure 1. Tegillarca granosa samples used for nitrite exposure. Figure 1. Tegillarca granosa samples used for nitrite exposure. 2.2. Preparation of ArtificialArtificial Seawater 2.2. Preparation of Artificial Seawater ArtificialArtificial seawater was used instead of natural seawater to minimize interference from other nutrientsArtificial present seawater in natural was seawaterseawaterused instead that wouldofwould natural aaffectffect seawater thethe studystudy to minimize findings.findings. ArtificialArtificialinterference seawaterseawater from other withwith salinitynutrients between present 25 in to natural 30 ppt seawater was prepared that would by dissolving affect the 30 study g of finefine findings. sea salt Artificial in 1 L of distilledseawater water with salinitythat was between heated to25 80 to °C30 toppt accelerate was prepared the dissolving by dissolving time, 30 and g of then fine cooled sea salt to in 28 1– L35 of °C. distilled Other water that was heated to 80 ◦C to accelerate the dissolving time, and then cooled to 28–35 ◦C. Other water parametersthat was heated were to monitored 80 °C to accelerate and controlledcontro thelled dissolving according time, to their and naturalthen cooled habitat,habitat to ,28 as– 35 shown °C. Other in Figure water2 2 andparameters as inin thethe were OrganizationOrganization monitored forfor and EconomicEconomic controlled Co-operationCo according-operation to andand their DevelopmentDevelopment natural habitat [[22]22]., .as TheThe shown oxygenoxygen in Figure contentcontent 2 andwas asmonitored in the Organization at a level of forleast Economic 90% concentration Co-operation (8.26 and–7.43 Development mg O2/L at [22]25 °C). The after oxygen aeration content and was monitored at a level of least 90% concentration (8.26–7.43 mg O2/L at 25 ◦C) after aeration and stabilizationwas monitored for at 2424 a hh level [[23]23].. of least 90% concentration (8.26–7.43 mg O2/L at 25 °C) after aeration and stabilization for 24 h [23]. 100 100 75 75 50 50 R² = 0.992 Mortality (%) Mortality 25 R² = 0.992 Mortality (%) Mortality 25 0 0 0 0.5 1 1.5 2 2.5 0 Exposure0.5 Concentration1 1.5 (mg/L)2 2.5 Exposure Concentration (mg/L) Figure 2. Mortality (%) of Tegillarca granosa after 72 h of exposure to a series of nitrite (NO2-N) Figure 2. Mortality (%) of Tegillarca granosa after 72 h of exposure to a series of nitrite (NO2-N) concentrations. Figureconcentrations. 2. Mortality (%) of Tegillarca granosa after 72 h of exposure to a series of nitrite (NO2-N) 2.3. Preparationconcentrations. of Nitrite Stock Solution 2.3. Preparation of Nitrite Stock Solution Stock solution of nitrite with concentration 100 mg/L was prepared by dissolving 0.15 g dried 2.3. Preparation of Nitrite Stock Solution sodiumStock nitrite solution (NaNO of 2nitrite) in artificial with concentration seawater and 100 bringing mg/L towas a volume prepared of 1000by dissolving mL. Before 0.15 preparing g dried thesodium stockStock nitrite solution, solution (NaNO NaNOof 2nitrite) in2 artificialpowder with concentration seawater was dried and at 105100bringing ◦mg/LC and towas kepta volume prepared in a desiccator of 1000by dissolving mL. prior Before to 0.15 utilization, preparing g dried insodiumthe order stock nitrite to solution, remove (NaNO NaNO any2) excess in2 powderartificial moisture was seawater dried that would andat 105 bringing a°Cffect and the tokept result.a volume in a Thedesiccator of stock 1000 solution mL.prior Before to wasutilization preparing prepared, in dailytheorder stock accordingto removesolution, toany NaNO the exces Standard2 powders moisture Method was that dried forwould at the 105 Examinationaffect °C and the kept result.