Environmental Homeopathy: Homeopathic Potencies Regulate the Toxicity and Growth of Raphidiopsis raciborskii (cyanobacteria) and can be Tracked Physico-Chemically. Part 1: Biological Results.

INTRODUCTION: Cyanobacteria are microorganisms found in many parts of the world and several genera, such as Raphidiopsis raciborskii, are producers of cyanotoxins. Homeopathic potencies have been found to modulate toxicity in different biological models, and the present study endeavors to discover whether this might also be the case with cyanobacteria. OBJECTIVES: Our objective was to investigate the possible effects of homeopathic potencies on the resilience of Artemia franciscana (brine shrimp) embryos to saxitoxin (STX; cyanotoxin) and on controlling the growth of R. raciborskii in vitro. METHOD: A. franciscana cysts were cultivated in seawater in 96-well plates to evaluate the hatching rate and vitality, plus the gene expression of heat shock proteins (HSPs), after being challenged with R. raciborskii extract containing 2.5 µg/L of STX and treated with different homeopathic potencies. Untreated wells were used as controls ("base-line"). Potencies were chosen from a screening process based on seven selected homeopathic preparations according to the similitude of STX symptoms (Sulphur, Zincum metallicum, Nitric acidum, Plumbum metallicum, Mercurius solubilis, Phosphoric acidum, Isotherapic from R. raciborskii extract; all at 6cH, 30cH and 200cH). Cultures of R. raciborskii maintained in an artificial seawater medium were equally treated with screened homeopathic potencies selected from the same list but specifically for their growth control as a function of time. RESULTS: A 15% lower rate of hatching of A. franciscana cysts was observed after treatment with Nitric acidum 6cH in comparison with baseline (p = 0.05). A complete toxicity reversal was seen after treatment with Isotherapic 200cH, with a 23-fold increase of Hsp 26 gene expression (p = 0.023) and a 24-fold increase of p26 gene expression (p ≤ 0.001) in relation to baseline. Nitric acidum 200cH and Mercurius solubilis 30cH limited the exponential growth of cyanobacteria up to 95% and 85% respectively (p ≤ 0.003) in relation to baseline. Succussed water presented only a transitory 50% inhibition effect. CONCLUSION: Isotherapic 200cH improved A. franciscana bioresilience to STX; Nitric acidum 200cH and Mercurius solubilis 30cH showed the optimal performance on limiting R. raciborskii growth. The results point to the potential of homeopathic potencies to mitigate environmental problems related to water quality.

Environmental Homeopathy: Homeopathic Potencies Regulate the Growth and Toxicity of Raphidiopsis raciborskii (cyanobacteria) and Can be Tracked Physico-Chemically. Part 2: Physico-chemical Results.

INTRODUCTION: The control of cyanobacterial toxicity and growth by homeopathic potencies was described in Part 1 of this two-part report. Here, a parallel approach characterized the physico-chemical features of the potencies used and the liquid media treated with them, correlating these results with their respective biological effects. OBJECTIVES: Our objective was to establish if physico-chemical parameters can track homeopathic potencies in seawater or artificial seawater medium (ASM)-1 and to discover whether these parameters correlate with previously described biological effects. METHOD: Artemia franciscana (brine shrimp) cysts were cultivated in seawater challenged with Raphidiopsis raciborskii extract and treated with different homeopathic potencies chosen from a screening process. Cultures of R. raciborskii maintained in ASM-1 were also treated with previously screened homeopathic potencies, and their growth was monitored as a function of time. The physico-chemical properties of the treated media (seawater or ASM-1) were evaluated by their interaction with solvatochromic dyes and changes in pH, conductivity and temperature. RESULTS: Coumarin 7 was found to be a marker for Nitric acidum 6cH and Isotherapic (R. raciborskii extract) 200cH in seawater (analysis of variance [ANOVA], p = 0.0015). Nile red was found to be a marker for Nitric acidum 200cH and Mercurius solubilis 30cH in ASM-1 (ANOVA, p ≤ 0.001). An increase in pH of ASM-1 and endothermic effects were observed after these treatments (two-way ANOVA, p = 0.0001). Seawater and ASM-1 to which potencies had been added were also subjected to a constant unidirectional 2,400 Gauss static magnetic field and found to have enhanced effects on the solvatochromic dyes tested. CONCLUSION: Homeopathic potencies were specifically traceable in aqueous media using solvatochromic dyes, especially when the samples were subjected to a magnetic field. Results from monitoring other physical parameters, such as pH and temperature, were less specific in relation to potency tracking. However, potency-induced endothermic effects might provide valuable thermodynamic data relating to the nature of potencies.

Highly Diluted Glyphosate Mitigates Its Effects on Artemia salina: Physicochemical Implications.

Glyphosate is an herbicide widely used in agriculture but can present chronic toxicity in low concentrations. Artemia salina is a common bio-indicator of ecotoxicity; it was used herein as a model to evaluate the effect of highly diluted-succussed glyphosate (potentized glyphosate) in glyphosate-based herbicide (GBH) exposed living systems. Artemia salina cysts were kept in artificial seawater with 0.02% glyphosate (corresponding to 10% lethal concentration or LC10) under constant oxygenation, luminosity, and controlled temperature, to promote hatching in 48 h. Cysts were treated with 1% (v/v) potentized glyphosate in different dilution levels (Gly 6 cH, 30 cH, 200 cH) prepared the day before according to homeopathic techniques, using GBH from the same batch. Controls were unchallenged cysts, and cysts treated with succussed water or potentized vehicle. After 48 h, the number of born nauplii per 100 µL, nauplii vitality, and morphology were evaluated. The remaining seawater was used for physicochemical analyses using solvatochromic dyes. In a second set of experiments, Gly 6 cH treated cysts were observed under different degrees of salinity (50 to 100% seawater) and GBH concentrations (zero to LC 50); hatching and nauplii activity were recorded and analyzed using the ImageJ 1.52, plug-in Trackmate. The treatments were performed blind, and the codes were revealed after statistical analysis. Gly 6 cH increased nauplii vitality (p = 0.01) and improved the healthy/defective nauplii ratio (p = 0.005) but delayed hatching (p = 0.02). Overall, these results suggest Gly 6cH treatment promotes the emergence of the more GBH-resistant phenotype in the nauplii population. Also, Gly 6cH delays hatching, another useful survival mechanism in the presence of stress. Hatching arrest was most marked in 80% seawater when exposed to glyphosate at LC10. Water samples treated with Gly 6 cH showed specific interactions with solvatochromic dyes, mainly Coumarin 7, such that it appears to be a potential physicochemical marker for Gly 6 cH. In short, Gly 6 cH treatment appears to protect the Artemia salina population exposed to GBH at low concentrations.

Environmental parameters modulate lead chloride toxicity on Artemia salina

Different environmental conditions can influence the effects of toxic agents on living beings. Recently, a series of experiments performed in Artemia salina submitted to different kinds of intoxication have shown that both, isotherapic and succussed watercan change Artemia salina ́s bio resilience at different levels. Moreover, it seems to vary according to the circalunar cycle. Objective:To verify if circalunar phases and water agitation can modify the toxicity of lead chloride on Artemia salina in vitro. Methodology:Artemia salina cysts were exposed to seawater containing 0.04% of lead chloride (equal to EC10 or 10% effective concentration, previously determined in a pilot study) in 96-well culture plates. Thirty-six experimental repetitions were performed in four series to observe the possible effects of adding stirred water, the so-called succussed water, and the moon phases. The hatched cysts were recorded after 48 hours using a digital microscope (1000x magnification) to identify the hatching percentage and the viability and mobility of the born nauplii. Results:The exposition of cysts to PbCl2 led to an increase in the hatching rate, and it was more evident during the full moon (p = 0.00014) The addition of succussed water into the seawater medium reduced this effect to the baseline levels. An increase in mobility was seen in nauplii born from exposed cysts during the full moon (p = 0.00077), but this effect was not affected by the treatment with succussed water. Discussion:Although the effects of lead chloride EC10 on the increase of nauplii hatching were expected, two environmental variables changed the sensitivity of cysts to this harmful stimulus. The circalunar cycle varied the hatching rate according to the moon phase, even in laboratory conditions, and the addition of succussed water into the medium reduced the hatching rate, even with different intensities according to the moon phase. The organization of nano and microbubbles generated after the succussion of water could be related to this protective effect and can explain, at least partially the effects of high diluted preparations on this biological context. Conclusion:Environmental factors, such as the circalunar cycle and products of water agitation, can modulate the adaptative control of hatching in Artemia salina exposed to lead chloride at EC10.

Effects of homeopathic preparations on raphidiopsis raciborskii (Cyanobacteria) toxicity in artemia salina mode

Cyanobacteria are microorganisms found in different parts of the world. Some genera are cyanotoxins producers a sodium channel blockingneurotoxin (saxitoxins). Some homeopathic preparations have been identified as remedial action on toxicity models in Artemia salina. This study aimed to observe whether homeopathic products influence the toxicity ofR.raciborskiiextract onA.salinaby inducing cyst hatching arrest, anembryo bioresiliencemodel previously developed in our laboratory (Pinto et al., 2021; Mohammad et al., 2022). Thus, previous toxicity testswere carried out on cysts in 96-well plates, using different concentrations of the extract obtained from regular cultivation of R. raciborskii in HCl 0.05M, whose strain, named T3, is kept in the laboratory of Cyanobacteria at FURGS, Brazil.The standardization of toxin concentration was based on an established scale developed at FURGS, in which the number of T3 filaments is associated withspecific saxitoxin concentrationsdefined by chromatography. The concentration of 2.6 µg/L was chosen since it reducedthe cysthatching rate by 30%, the ideal level to observe embryo bioresilience. Then, a screeningstudy with 22 homeopathic preparations was tested blind in three experimental series, in duplicate,against threecontrols (unchallenged, water,and succussed water)for possible toxicity attenuationon Artemia salinacysts hatching rate. Homeopathic medicines were prepared in pure,sterile water from a stock homeopathic solution, one potency below the working potency. After the 1:100 dilution, 100 succussions were made using a robotic arm (Denise, Autic). The medicines were inserted into the seawater on a 10% basis. Due to the high sensitivity of A. salinato the circalunar variations, all experiments were performed during the first quarter moon. Statistical analysis was performed by two-way ANOVA followed by Tukey, with α=0.05. The most significant results indicative of bioresilience improvement were seen after the treatment with Nitric acidum6 cH, Plumbum metallicum6 cH, isotherapic 200 cH, and hydrochloric acid 1 cH being the last one used as a vehicle of the extracts. Thus, these preparations were chosen to be used in further experiments. In conclusion, the Artemia salinamodel has also beenuseful to study bioresilienceimprovement by homeopathic medicines after intoxication with saxitoxin.

Mercury chloride toxicity attenuation of the Brine Shrimp Artemia Salinaafter treatment with Mercurius corrosivusas isotherapic

Isotherapics preparedfromtoxic substances have been described as attenuation factors for heavy metal intoxicationin aquatic animals. Herein, Artemia salinaand mercury chloride were usedas a model to identify treatment-related bioresilience. The aim was to describe the effects of Mercurius corrosivus(MC) in different potencies on Artemia salinacyst hatching and on mercury bioavailability. Artemia salinacysts were exposed to 5.0 µg/mL of mercury chloride during the hatching phase. MC6cH, 30cH, and 200cHwere prepared and poured into artificial seawater. Different controls were used (nonchallenged cysts and challenged cysts treated with water, succussed water, and Ethilicum 1cH). Four series of nine experiments were performed for4 weeks to evaluate the percentage of cyst hatchingconsidering all moon phases. Soluble total mercury (THg) levels and precipitated mercury content were also evaluated. Solvatochromic dyes were used to check for eventual physicochemical markers of MCbiological activity. Two-way analysis of variance (ANOVA) with mixed modelswas used for evaluating the effect of different treatments andthe simultaneous influence of the moon phases on the cystshatching rate, at both observation times (24 and 48 hours).When necessary, outliers were removed, using the Tukeycriterion.Thelevel of significance αwas set at 5%. Significant delay (p<0.0001) in cyst hatching was observed after treatment with MC30cH, compared with the controls. An increase inTHg concentration in seawater (p<0.0018) and of chlorine/oxygen ratio (p<0.0001) in suspended micro-aggregateswas also seen, with possiblerelation with mercury bioavailability. Specific interaction of MC30cH with the solvatochromic dye ET33 (p<0.0017) was found. The other observed potencies of Mercurius corrosivus6 and 200 cH were not significant in relation to the observed groups.The results werepostulated as being protective effects of MC30cH on Artemia salina, by improving its bioresilience.

Bioresilience to Mercury Chloride of the Brine Shrimp Artemia Salina after Treatment with Homeopathic Mercurius Corrosivus.

INTRODUCTION: Finding solutions to mitigate the impact of pollution on living systems is a matter of great interest. Homeopathic preparations of toxic substances have been described in the literature as attenuation factors for intoxication. Herein, an experimental study using Artemia salina and mercury chloride was developed as a model to identify aspects related to bioresilience. AIMS: The aim of the study was to describe the effects of homeopathic Mercurius corrosivus (MC) on Artemia salina cysts hatching and on mercury bioavailability. METHODS: Artemia salina cysts were exposed to 5.0 µg/mL of mercury chloride during the hatching phase. MC potencies (6cH, 30cH, and 200cH) were prepared in sterile purified water and poured into artificial sea water. Different controls were used (non-challenged cysts and challenged cysts treated with water, succussed water, and Ethilicum 1cH). Four series of nine experiments were performed to evaluate the percentage of cyst hatching. Soluble total mercury (THg) levels and precipitated mercury content were also evaluated. Solvatochromic dyes were used to check for eventual physicochemical markers of MC biological activity. RESULTS: Significant delay (p < 0.0001) in cyst hatching was observed only after treatment with MC 30cH, compared with controls. This result was associated with an increase of THg concentration in water (p = 0.0018) and of chlorine/oxygen ratio (p < 0.0001) in suspended micraggregates, suggesting changes in mercury bioavailability. A specific interaction of MC 30cH with the solvatochromic dye ET33 (p = 0.0017) was found. CONCLUSION: Changes in hatching rate and possible changes in Hg bioavailability are postulated as protective effects of MC 30cH on Artemia salina, by improving its natural bioresilience processes.