Ammonium chloride

Ortho-Phosphoric acid (85–88 %) and sodium hydroxide (99 %) were purchased from R & M Chemicals. Ammonium chloride (≥99.5 %), sodium nitrite (≥97 %) and sodium nitrate (≥99.0 %) were purchased from Sigma. All chemicals were of analytical grade and used as received without further purification. Distilled water was used in all experiments. Green mango leaves, used coffee grounds, and red cabbages were sourced from apple mango trees, local coffee shops, and local markets in Kuala Nerus, Terengganu, respectively, during April and June 2024.

An investigation was conducted to determine the LC₅₀ using a static method by refreshing the water every 24 h and substituting the required levels of concentration. We implemented two separate concentrations of ammonium chloride (Merck, Germany) in our methodology, which resulted in a mortality rate of 100% and no losses [50 (link)]. Following this, six concentrations of ammonium chloride were determined exponentially within the ranges of the two preceding doses. Subsequently, following the period of adjustment, the fish were segregated into six distinct groups, each comprising 10 fish [53 (link)]. The fish underwent a 96-h examination period, during which the mortality rate was documented at 24-h intervals [54 (link)]. Throughout the duration of the experiment, both the temperature and pH were maintained at a constant level. The LC₅₀ was determined at 24, 48, 72, and 96 h using probit software following the recording of mortality [22 (link)].

T4 was prepared as described by Pierres and Gaugain-Hamidi [14] with slight modification to increase both the solubility and stability of T4 preparation. Briefly, each 100 ml of the preparation contained all of the following: (Sodium-L-thyroxin: 2 mg, NaHCO₃: 0.336 g, hydroxypropyl-β-cyclodextrin: 2 g, EDTA: 0.1 g, ammonium chloride: 0.5 g, ethanol: 15 ml, all obtained from Sigma), pH was adjusted to 8.5, then completed to 100 ml with double distilled water and stored at 4 C°.
Animals were divided into 6 groups based on genetic background and treatment (n = 8/group) as follows: wild-type; wild-type + T4; ZmRacD; ZmRacD + T4; ZmRacD + carvedilol + T4; ZmRacD + pravastatin + T4. The study duration was extended to 3 weeks instead of 2 weeks [12] (link) based on previous studies demonstrating that the largest part (70–80%) of the myocardial remodeling process in rodent e.g. rat occurs within the first 3 weeks [15] (link). Basal echocardiography parameters were determined before starting T4 treatment and on a weekly basis thereafter, till the experiment was terminated. T4 (200 µg/Kg) was injected intraperitoneally as previously described [12] (link).
carvedilol (2 mg/Kg) or pravastatin (10 mg/Kg) were administered by intraperitoneal injection 1 hour before T4 administration. carvedilol was prepared as reported in [16] (link) with some modifications. Briefly, carvedilol from Sigma was dissolved in a minimal amount of DMSO, and then diluted with 5% dextrose solution containing 1–2 drops of glacial acetic acid (final DMSO concentration ≤0.1%). The dose of carvedilol used in this study was based on previous studies that showed its effectiveness in a rat model of cardiac dilation and it is roughly equivalent to the carvedilol dose in human [17] (link). Pravastatin was prepared and injected as previously described [12] (link). At the end of the treatment period animals underwent echocardiography. Directly after echocardiography, animals were sacrificed, and hearts were excised and processed for further experiments.

The industrial cyanide-containing waste from the jewelry was supplied by Avenir S.L. and Gemasur S.L. (Córdoba, Spain). Ammonium chloride, sodium acetate, sodium malate, sodium citrate, and sodium octanoate were supplied by Sigma–Aldrich (St. Louis-MO, USA). Nitrogen was purchased from Air Liquid (Paris, France). All other chemicals used in the study were of analytical grade. Solutions were prepared by using Milli-Q water (Millipore, Bedford-MA, USA). Wastes containing cyanide or other toxic chemicals were handled and disposed by the Environmental Protection Unit, University of Córdoba.