Significance of Mg-hardness and fluoride in drinking water on chronic kidney disease of unknown etiology in Monaragala, Sri Lanka.

The epidemic of chronic kidney disease of unknown etiology (CKDu) that contributes significantly to morbidity and mortality rates among dry-zonal farming communities has become a public health priority in Sri Lanka. Though a large number of hypotheses were introduced as causative factors, none of them have been confirmed so far. As drinking water quality is among the most suspected causative factors for the emergence of CKDu, a detailed hydro-geochemical investigation was carried out concurrently with the population screening in the Monaragala district of Sri Lanka where high incidences of CKDu are reported. A population screening was performed selecting 46,754 people using both dipstick proteinuria test and Albumin-Creatinine Ratio (ACR). The results revealed that the disease prevalence is about 6.7 % in the district. A total of 60 groundwater samples, 30 each, were collected from CKDu-prevalent locations and control locations where there are no CKDu cases reported. The samples were analyzed to identify any possible linkage between water quality and disease prevalence. Concentrations of hardness, F-, Na+, and Mg2+ in groundwater revealed a statistically significant difference between CKDu and control wells at a confident level of p = 0.05. The study revealed that alkali (Na++K+) and alkaline earth cations (Mg2+, Ca2+, Sr2+, Ba2+) were relatively higher in drinking water sources used by CKDu patients, compared to the well waters used by healthy individuals. Nearly 87 % of the wells used by CKDu cases showed higher fluoride levels that exceed the threshold level (1.0 mg L-1). Contents of nephrotoxic trace elements such as As, Cd, and Pb were found to be comparable in both types of wells and were well below the WHO permissible levels, thus negating their prime influence on the CKDu prevalence. It is obtrusive that the elevated fluoride levels together with water hardness associated with higher Mg2+ levels have a possible relation with CKDu and may influence the disease progression.

Water hardness alleviates the stress response caused by waterborne zinc in goldfish Carassius auratus.

In this study, the combined effect of waterborne Zn and water hardness on the stress response in the goldfish Carassius auratus was investigated. Goldfish were exposed to Zn concentrations of 0.5, 1.0, and 3.0 mg/L and water hardness of 90, 270, and 450 mg/L CaCO3 for 1, 3, 7, and 14 d. After exposure, it was determined that higher the Zn concentration, the more obvious the stress response. However, the stress response reduced with increasing water hardness. An increase in the Zn concentration caused stress responses in fish according to the increase in the mRNA expressions of corticotropin-releasing hormone and adrenocorticotropic hormone and cortisol level in the hypothalamus-pituitary-interrenal axis. The expression of these factors was the highest on day 7 and decreased on day 14. Furthermore, to evaluate the stress change in the liver tissue, we analyzed alanine aminotransferase, aspartate aminotransferase, and heat shock protein 70 concentrations to determine the damage caused by Zn and the change in water hardness. Immunohistochemistry staining for Na+/K+-ATPase in the gills showed that the gill activity was inhibited by Zn, and an increase in water hardness could improve Na+/K+-ATPase. In conclusion, we found that increasing water hardness is a successful method to reduce the stress response in goldfish caused by Zn.

Coping strategies in response to different levels of elevated water hardness in channel catfish (Ictalurus punctatus): Insight into ion-regulatory and histopathological modulations.

Water hardness above the optimal level can incite toxic effects in fish, which are often species specific. Hence, we aimed at obtaining insights on the potential effects of elevated water hardness as well as coping strategies in channel catfish (Ictalurus punctatus). First, a toxicity assay was performed where the 96 h-LC50 was calculated as 4939 mg/L CaCO3. Thereafter, to gain knowledge on the underlying adaptive strategies to high water hardness, fish were exposed to seven hardness levels (150, 600, 1000, 1500, 2000, 3000 and 4000 mg/L CaCO3 at pH 8.15) for 15 days. Results showed that branchial activities of Ca2+-ATPase and Na+/K+-ATPase, which facilitate Ca2+ uptake, reduced starting respectively from 1000 mg/L and 1500 mg/L CaCO3. Nevertheless, Ca2+ burden in plasma and tissue (gills, liver and intestine) remained elevated. Hardness exposure also disturbed cations (Na+, K+, Mg2+) and minerals (iron and phosphorus) homeostasis in a tissue-specific and dose-dependent manner. Both hemoglobin content and hematocrit dropped significantly at 3000-4000 mg/L CaCO3, with a parallel decline in iron content in plasma and gills. Muscle water content rose dramatically at 4000 mg/L CaCO3, indicating an osmo-regulation disruption. Higher hardness of 3000-4000 mg/L CaCO3 also incited a series of histopathological modifications in gills, liver and intestine; most likely due to excess Ca2+ accumulation. Overall, these data suggest that channel catfish can adapt to a wide range of elevated hardness by modulating Ca2+ regulatory pathways and histomorphological alterations, however, 1500 mg/L CaCO3 and above can impair the performance of this species.

Development of regional water quality criteria of lead for protecting aquatic organism in Taihu Lake, China.

Lead is a widely distributed priority controlled heavy metals in aquatic system, its toxicity to aquatic organisms affected by water quality parameters. This study investigated the acute toxicity of lead (Pb) to the aquatic organisms in Taihu Lake under various water hardness, corresponding regional water quality criteria were derived. The acute toxicity experimental results revealed that the toxicity of Pb to aquatic organisms increased with water hardness. The Pb toxicity has a highest toxicity at water hardness 50 mg/L (expressed as CaCO3), especially for Palaemon modestus where the 96 h LC50 value was 0.024 mg/L. The hazardous concentration for 5% of biological species (HC5) values were determined via species sensitivity distribution (SSD) method as 94.0 µg/L, 222.3 µg/L and 375.8 µg/L for Pb at water hardness 50, 150, and 250 mg/L, respectively. The assessment factor (AF) value was set at 2, followed by the current SSD framework where European commission recommend a fixed AF of 5-1. Thus, the predicted no effect concentration (PNEC) values was 47.0 µg/L, 111.2 µg/Land 187.9 µg/L at water hardness 50, 150, and 250 mg/L, respectively. Meanwhile, the short-term water quality criteria of Pb for Taihu lake aquatic organisms were derived as 111.2 µg/L at water hardness 150 mg CaCO3/L. The long-term water quality criteria were derived as 4.3 µg/L by using acute/chronic ratio 51.29. When the derived value was used for Taihu Lake, 2.7% of the sampling sites in Taihu Lake was exceeded this criterion. The results of this study can provide technical methods and basic information for deriving Pb regional water quality criteria for protecting native aquatic organisms, in China.

Photodynamic Inactivation of Legionella pneumophila Biofilm Formation by Cationic Tetra- and Tripyridylporphyrins in Waters of Different Hardness.

The bacterium Legionella pneumophila is still one of the probable causes of waterborne diseases, causing serious respiratory illnesses. In the aquatic systems, L. pneumophila exists inside free-living amoebae or can form biofilms. Currently developed disinfection methods are not sufficient for complete eradication of L. pneumophila biofilms in water systems of interest. Photodynamic inactivation (PDI) is a method that results in an antimicrobial effect by using a combination of light and a photosensitizer (PS). In this work, the effect of PDI in waters of natural origin and of different hardness, as a treatment against L. pneumophila biofilm, was investigated. Three cationic tripyridylporphyrins, which were previously described as efficient agents against L. pneumophila alone, were used as PSs. We studied how differences in water hardness affect the PSs' stability, the production of singlet oxygen, and the PDI activity on L. pneumophila adhesion and biofilm formation and in biofilm destruction. Amphiphilic porphyrin showed a stronger tendency for aggregation in hard and soft water, but its production of singlet oxygen was higher in comparison to tri- and tetracationic hydrophilic porphyrins that were stable in all water samples. All three studied porphyrins were shown to be effective as PDI agents against the adhesion of the L. pneumophila to polystyrene, against biofilm formation, and in the destruction of the formed biofilm, in their micromolar concentrations. However, a higher number of dissolved ions, i.e., water hardness, generally reduced somewhat the PDI activity of all the porphyrins at all tested biofilm growth stages.

Derivation of water quality criteria of zinc to protect aquatic life in Taihu Lake and the associated risk assessment.

Zinc is a widely distributed environmental pollutants and has been listed as priority heavy metal pollutant in China. Similar as other heavy metals, toxicity of zinc to aquatic organisms affects by environmental factors such as water hardness. It is necessary to develop regional water quality criteria (WQC) to protect native aquatic life against zinc due to the diversity of aquatic organisms' variability across different water systems, as a concretization and supplement for national zinc WQC. This study derived WQC for zinc by species sensitivity distribution (SSD) curve method. The zinc toxicity data of the aquatic organisms in Taihu Lake used in SSD curve was collected based on published toxicity data for zinc with hardness values and supplemented with acute toxicity tests conducted in this study. Six aquatic organism natives to Taihu Lake were selected to conduct zinc acute toxicity test in a range of hardness conditions. The relationship between water hardness and zinc toxicity was constructed. The criterion maximum concentration (CMC) and criterion continuous concentration (CCC) for zinc in Taihu Lake were then derived, which considered the water quality and taxonomic groups in Taihu Lake. The CMC and CCC were 100.69 µg/L and 30.79 µg/L, respectively. The environmental risk of zinc to Taihu Lake are acceptable, at moderate to low levels. This study has provided a basis for regional water quality criterion derivation and risk assessment in China.

Effect of Water Hardness on Catechin and Caffeine Content in Green Tea Infusions.

The health benefits of green tea are associated with its high catechin content. In scientific studies, green tea is often prepared with deionized water. However, casual consumers will simply use their local tap water, which differs in alkalinity and mineral content depending on the region. To assess the effect of water hardness on catechin and caffeine content, green tea infusions were prepared with synthetic freshwater in five different hardness levels, a sodium bicarbonate solution, a mineral salt solution, and deionized water. HPLC analysis was performed with a superficially porous pentafluorophenyl column. As water hardness increased, total catechin yield decreased. This was mostly due to the autoxidation of epigallocatechin (EGC) and epigallocatechin gallate (EGCG). Epicatechin (EC), epicatechin gallate (ECG), and caffeine showed greater chemical stability. Autoxidation was promoted by alkaline conditions and resulted in the browning of the green tea infusions. High levels of alkaline sodium bicarbonate found in hard water can render some tap waters unsuitable for green tea preparation.

The Role of Environmental Exposures in Atopic Dermatitis.

PURPOSE OF REVIEW: Although genetic factors clearly play a role in the development of atopic dermatitis (AD), the recent dramatic increase in the prevalence of AD in low- and middle-income countries is not consistent with only a role of genetic factors. These findings strongly suggest that environmental factors may play an important role in the pathogenesis of AD. RECENT FINDINGS: We reviewed the role of gene-environment studies; in utero exposures including tobacco smoke, alcohol, maternal stress, various digestive supplements, and gestational diabetes; early-life exposures including diet, gut microbiota, antibiotics, and breastfeeding; climate including temperature, ultraviolet radiation exposure, and air pollution; and household products, indoor allergens, water hardness, pH, and skin microbiota and their effects on AD. Environmental factors definitely play a role in the pathogenesis of AD. However, identifying definitive factors continues to be difficult in the setting of conflicting evidence and the complex interactions between genotypes and the environment resulting in a multitude of AD phenotypes. All of the different environmental interactions discussed highlight the importance of intervening on multiple levels in a patient's environment to improve or even prevent AD symptoms. Further, the importance of modifying environmental factors early on in a person's life is demonstrated. When possible, all of these environmental factors should be considered in treating a patient with AD and the appropriate modifications should be made at population and individual levels.

In situ, high-resolution measurement of labile phosphate in sediment porewater using the DET technique coupled with optimized imaging densitometry.

Obtaining two-dimensional distributions of reactive phosphorus in sediment porewater is very important for understanding fine-scale phosphorus mobilization and sequestration processes in sediments. In this study, the diffusive equilibrium in thin films (DET) measurement based on computer imaging densitometry (CID) was studied in detail with optimal conditions described. This study focuses on evaluating the two-dimensional colorimetric DET method coupled with CID (DET-CID method) for porewater labile phosphate measurements. The result shows that the red channel filter is the optimum channel for sensitivity to process the image. Additionally, staining time and temperature have great influence on the method, and 20 min staining time and ≥25 °C staining temperature were recommended. The minimum detection limit of labile phosphate of this method was 0.300 mg P/L, and the maximum detection limit could reach 50.00 mg P/L. The DET-CID technique can be used to measure labile phosphate in a wide range of acidic and alkaline water bodies (pH = 2-10 and water hardness from 0 to 2000 mg/L as CaCO3). The linear regression analysis shows that this technique presents very similar results compared with other two existing methods (R2 = 0.999). Our results would give insights into the precisely measurements of labile phosphate in field applications.

Experimental rat model for acute tubular injury induced by high water hardness and high water fluoride: efficacy of primary preventive intervention by distilled water administration.

BACKGROUND: High water hardness associated with high water fluoride and the geographical distribution of Chronic Kidney Disease of unknown etiology (CKDu) in Sri Lanka are well correlated. We undertook this study to observe the effects of high water hardness with high fluoride on kidney and liver in rats and efficacy of distilled water in reducing the effects. METHODS: Test water sample with high water hardness and high fluoride was collected from Mihinthale region and normal water samples were collected from Kandy region. Twenty-four rats were randomly divided into 8 groups and water samples were introduced as follows as daily water supply. Four groups received normal water for 60 (N1) and 90 (N2) days and test water for 60 (T1) and 90 (T2) days. Other four groups received normal (N3) and test (T3) water for 60 days and followed by distilled water for additional 60 days and normal (N4) and test (T4) water for 90 days followed by distilled water for another 90 days. The rats were sacrificed following treatment. Serum samples were subjected to biochemical tests; serum creatinine, urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and elemental analysis. Histopathological examinations were carried out using kidney and liver samples. RESULTS: Test water treated groups were associated with acute tubular injury with loss of brush border and test water followed with distilled water treated groups maintained a better morphology with minimal loss of brush border. Serum creatinine levels in T1 and T2 groups and urea level in T2 group were significantly (p < 0.05) increased compared to control groups. After administration of distilled water, both parameters were significantly reduced in T4 group (p < 0.05) compared to T2. Serum AST activity was increased in T4 group (p < 0.05) compared to control group with no histopathological changes in liver tissues. The serum sodium levels were found to be much higher compared to the other electrolytes in test groups. CONCLUSION: Hard water with high fluoride content resulted in acute tubular injury with a significant increase in serum levels of creatinine, urea and AST activity. These alterations were minimized by administering distilled water.

Regulations for calcium, magnesium or hardness in drinking water in the European Union member states.

Very low or very high concentrations of calcium (Ca) and magnesium (Mg) or total hardness in drinking water have been empirically recognized as the cause of the problems with corrosion, scaling, or taste of water. A large body of scientific evidence over the last 60 years has also attributed health problems to both extremes of these minerals in water. For example, drinking water low in Mg significantly increases the likelihood of cardiovascular mortality. This knowledge is not properly considered in the drinking water quality regulations, especially as more and more water is now produced by desalination disturbing its natural mineral composition. Although the current EU Drinking Water Directive does not include limits for hardness, Ca or Mg, most member states regulate these parameters either through law (12 countries) or technical guidance (7 countries), and several countries have issued educational information for the public. However, the regulations in most countries are not based on current research and not comparable because some provide a recommended range while others provide mandatory minimum or maximum limit values. This summary of the situation in 28 European Union countries is intended to provide information that can contribute to the implementation of the revised EU Directive.

Bioluminescent Ratiometric Indicator for Analysis of Water Hardness in Household Water.

Water hardness (WH) is a useful parameter for testing household water, such as drinking, cooking, and washing water. Many countries around the world use pipeline water in their houses, but there is a need to monitor the WH because hard water has a negative impact on appliances. Currently, WH is often measured using chemical dye-based WH indicators, and these techniques require expensive equipment, and trained personnel. Therefore, a low-cost and simple measurement method has been desired. Here, we report LOTUS-W, which consists of a luciferase, Nanoluc, a yellow fluorescent protein Venus, and a Ca2+/Mg2+ detection domain of human centrin 3. The binding of Ca2+/Mg2+ to this indicator changes the conformation of human centrin 3, and induces bioluminescence resonance energy transfer (BRET) from Nanoluc to Venus, which changes its emission spectrum about 140%. The dissociation constants of LOTUS-W for Ca2+/Mg2+ are approximately several mM, making it suitable for measuring WH in the household water. With this indicator in combination with a smartphone, we have demonstrated that it is possible to evaluate WH easily and quickly. This novel indicator has the potential to be used for measuring not only household water but also water used in the food industry, etc.

Transcriptome analysis of the fish pathogen Flavobacterium columnare in biofilm suggests calcium role in pathogenesis.

BACKGROUND: Flavobacterium columnare is the causative agent of columnaris disease that affects cultured freshwater fishes worldwide. F. columnare easily colonizes surfaces by forming biofilm, which helps the pathogen resist antibiotic and disinfectant treatments. Previously, we had shown that increasing concentrations of calcium (Ca2+) promoted biofilm formation by F. columnare. The objective of this study was to further characterize the role of Ca2+ on biofilm formation and to compare the transcriptome profiles of planktonic and biofilm cells. RESULTS: RNA-Seq analysis was conducted to identify genes that were differentially expressed between the following states: i) planktonic cells in control medium (P), ii) planktonic cells in calcium-enriched medium (P/Ca), and iii) biofilm cells in calcium-enriched medium (B/Ca). Overall, we identified 441 significant (FDR-adjusted p < 0.05, fold change > 2) differentially expressed genes (DEGs) between P and B/Ca samples; 112 significant DEGs between P/Ca and B/Ca samples, and 175 significant DEGs between P/Ca and P samples, corresponding to 15.87, 4.03 and 6.30% of the total protein-coding sequences, respectively. The significant DEGs fell into different functional categories including iron acquisition, oxidative stress response, extracellular protein secretion, and respiratory metabolism. CONCLUSIONS: Our results posit Ca2+ as a critical signal in regulating bacterial surface adhesion and biofilm formation in F. columnare. Living in biofilm elicited a shift in several metabolic pathways that allowed the cells to cope with oxidative stress and nutrient starvation. In addition, Ca2+ supplementation induced the expression of putative virulence factors in F. columnare, such as extracellular protein secretion and iron acquisition.

Association of consumption of excess hard water, body mass index and waist circumference with risk of hypertension in individuals living in hard and soft water areas.

Chronic exposure to soft drinking water increases the risk of hypertension. We conducted a cross-sectional study in 2017 in two study areas in the Poldasht County to assess the relation of hardness, body mass index and waist circumference with risk of hypertension. Total water hardness was measured by gravimetric methods. Hardness of > 180 ppm was considered to be hard drinking water. The Shiblu and Gharghologh areas had a four times higher mean total hardness level in drinking water (968.69 mg/L as CaCO3) than the Sarisoo and Agh Otlogh (180 mg/L as CaCO3) areas. According to the results of the study, the prehypertension prevalence in areas with high and low water hardness was calculated as 23.15% (18.11-29.15) and 46.84% (37.61-57.64), respectively. Moreover, the result showed the prevalence of hypertension and prehypertension was higher in regions with low hardness than those with high hardness, which was statistically significant (P < 0.001). The results of logistic regression showed that age, body mass index, waist circumference and hardness of drinking water were significantly related to hypertension. Accordingly, hypertension was low in people of regions with high hardness (OR 0.26, 0.17-0.42). This report examined whether total hardness in drinking water was protective against hypertension. This is an important finding for the ministry of human health as well as for the water and sewage company.

Prevalence of atopic dermatitis in infants by domestic water hardness and season of birth: Cohort study.

BACKGROUND: Atopic dermatitis (AD) appears to be more common in regions with hard domestic water and in children with a fall/winter birth. However, it is unknown whether a synergistic effect exists. OBJECTIVE: We sought to evaluate the association between domestic water hardness and season of birth, respectively, with onset of AD within the first 18 months of life in a large Danish birth cohort. METHODS: Of children from the Danish National Birth Cohort, 52,950 were included. History of physician-diagnosed AD and population characteristics were obtained from interviews. Birth data were obtained from the Civil Registration System, and domestic water hardness data were obtained from the Geological Survey of Denmark and Greenland. The relative prevalence (RP) of AD was calculated by using log-linear binomial regression. RESULTS: The prevalence of AD was 15.0% (7,942/52,950). The RP of AD was 5% (RPtrend, 1.05; 95% CI, 1.03-1.07) higher for each 5° increase in domestic water hardness (range, 6.60-35.90 German degrees of hardness [118-641 mg/L]). Although the RP of AD was higher in children with a fall (RP, 1.24; 95% CI, 1.17-1.31) or winter (RP, 1.18; 95% CI, 1.11-1.25) birth, no significant interaction was observed with domestic water hardness. The population attributable risk of hard domestic water on AD was 2%. CONCLUSION: We observed that early exposure to hard domestic water and a fall/winter birth was associated with an increase in the relative prevalence of AD within the first 18 months of life. Although the 2 exposures did not interact synergistically, a dose-response relationship was observed between domestic water hardness and AD.

[The evaluation of the impact of quality and microelement composition of drinking water on the incidence of urolithiasis in various regions of Dagestan].

AIM: to study the influence of qualitative and microelement composition of drinking water on the incidence of urolithiasis in different regions of the Republic of Dagestan. MATERIALS AND METHODS: The statistical data of the Ministry of Health of the Republic of Dagestan for 2000-2016 yy. were used. A total of 12123 outpatient and inpatient charts of patients with urinary stone disease were analyzed. The annual average quality of drinking water, as well as the contents of microelements in different climatic and geographical areas of the region were assessed. THE RESULTS: The high morbidity among the residents of the flat territory was established which was associated with the highest water hardness, high content of microelements and salts of heavy metals that exceeded the acceptable concentrations. The number of patients with urinary stone disease living in mountainous areas is much less (11.8%) than in the flat area (83.08%). A relationship between quality of water, microelement composition and urinary stone disease in this area was found. The stone disease was more common in women (66.3%) than in men (33.7%). CONCLUSION: Our results can be used for the prevention of urinary stone disease given that the Republic of Dagestan is an endemic region.

Association between domestic water hardness, chlorine, and atopic dermatitis risk in early life: A population-based cross-sectional study.

BACKGROUND: Domestic water hardness and chlorine have been suggested as important risk factors for atopic dermatitis (AD). OBJECTIVE: We sought to examine the link between domestic water calcium carbonate (CaCO3) and chlorine concentrations, skin barrier dysfunction (increased transepidermal water loss), and AD in infancy. METHODS: We recruited 1303 three-month-old infants from the general population and gathered data on domestic water CaCO3 (in milligrams per liter) and chlorine (Cl2; in milligrams per liter) concentrations from local water suppliers. At enrollment, infants were examined for AD and screened for filaggrin (FLG) skin barrier gene mutation status. Transepidermal water loss was measured on unaffected forearm skin. RESULTS: CaCO3 and chlorine levels were strongly correlated. A hybrid variable of greater than and less than median levels of CaCO3 and total chlorine was constructed: a baseline group of low CaCO3/low total chlorine (CaL/ClL), high CaCO3/low total chlorine (CaH/ClL), low CaCO3/high total chlorine (CaL/ClH) and high CaCO3/high total chlorine (CaH/ClH). Visible AD was more common in all 3 groups versus the baseline group: adjusted odds ratio (AOR) of 1.87 (95% CI, 1.25-2.80; P = .002) for the CaH/ClL group, AOR of 1.46 (95% CI, 0.97-2.21; P = .07) for the CaL/ClH, and AOR of 1.61 (95% CI, 1.09-2.38; P = .02) for the CaH/ClH group. The effect estimates were greater in children carrying FLG mutations, but formal interaction testing between water quality groups and filaggrin status was not statistically significant. CONCLUSIONS: High domestic water CaCO3 levels are associated with an increased risk of AD in infancy. The influence of increased total chlorine levels remains uncertain. An intervention trial is required to see whether installation of a domestic device to decrease CaCO3 levels around the time of birth can reduce this risk.

The Effect of Water Hardness on Mortality of Zebrafish (Danio rerio) During Exposure to Oxytetracycline.

Marking of fish otoliths with oxytetracycline and tetracycline is a widely used method to evaluate the effectiveness of stocking operations. Available protocols for the labeling of fish specify a number of factors influencing mark quality and potential risk for fish during marking. This study investigates the influence of water hardness on mortality of freshwater fish during marking with OTC. In order to pursue this question complexation of OTC with Mg(2+) and Ca(2+) cations was measured spectrophotometrically. Furthermore, zebrafish (Danio rerio) were immersed in OTC solutions (1200 mg/L; 48 h immersion) combined with varying levels of water hardness (5.5, 15.5, 25.5, 32.5°dH). The amount of OTC-Mg-Ca-complexes was positively correlated to water hardness. Moreover, it could be demonstrated that mortality of zebrafish during marking varied as a factor of water hardness. Highest mortalities occurred at the lowest (5.5°dH) and the highest (32.5°dH) tested levels during marking with OTC.

Effect of water hardness on the production and microbicidal efficacy of slightly acidic electrolyzed water.

Slightly acidic electrolyzed water (SAEW) has been proved as an effective sanitizer against microorganisms attached to foods. However, its physical properties and inactivation efficacy are affected by several factors such as water hardness. Therefore, in this study the effect of water hardness on SAEW properties were studied. Pure cultures of foodborne bacteria were used in vitro and in vivo to evaluate the inactivation efficacy of the SAEWs produced. Results obtained showed water hardness to be an important factor in the production of SAEW. Low water hardness may result in the necessity of further optimization of production process. In this study the addition of 5% HCl and 2 M NaCl at 1.5 mL/min flow rate was found to be the best electrolyte concentration for the optimization of SAEW production from low hardness water (34 ± 2 mg/L). Furthermore, the results showed that pre-heating was a better approach compared to post-production heating of SAEW, resulting in higher ACC values and therefor better sanitization efficacy.

Calcium-dependent behavioural responses to acute copper exposure in Oncorhynchus mykiss.

Using rainbow trout Oncorhynchus mykiss, the present study demonstrated that: (1) calcium (Ca) increased the range of copper (Cu) concentrations that O. mykiss avoided; (2) Ca conserved the maintenance of pre-exposure swimming activity during inescapable acute (10 min) Cu exposure. Data showed that when presented with a choice of Cu-contaminated water (ranging from 0 to 454 µg Cu l⁻¹ ) and uncontaminated water in a choice tank, O. mykiss acclimated and tested at low Ca concentration (3 mg Ca l⁻¹ avoided the 10 µg Cu l⁻¹ only. By contrast, O. mykiss acclimated and tested at high Ca concentration (158 mg Ca l⁻¹) avoided all the Cu concentrations ≥37 µg⁻¹. The Cu avoidance was connected with increased spontaneous swimming speed in the Cu-contaminated water. When subjected to inescapable Cu exposure (35 µg Cu l⁻¹), O. mykiss acclimated and tested at low Ca concentration reduced their spontaneous swimming speed, whereas no response was observed in O. mykiss acclimated and tested at high Ca concentration. Collectively, the data support the conclusion that in O. mykiss the behavioural responses to acute Cu exposure are Ca-dependent.