244 Original Research Article

Bioresilience to Mercury Chloride of the Brine

Shrimp Artemia Salina after Treatment with
Homeopathic Mercurius Corrosivus
Andreia Adelaide G. Pinto1 Mirian Y. de Oliveira Nagai1 Ednar Nascimento Coimbra1
Suham Nowrooz Mohammad2 Jefferson Souza Silva1 Adalberto Von Ancken1
Sandra Augusta G. Pinto1 Michelle Sanchez Aguiar1 Maristela Dutra-Correa1
Marcos Antonio Hortellani2 Adriana Miranda2 Jorge Eduardo de Souza Sarkis2
Ivana Barbosa Suffredini1 Giovani Bravin Peres1 Maria Martha Bernardi1 Steven John Cartwright3
Leoni Villano Bonamin1

1 Graduation Program in Environmental and Experimental Pathology, Address for correspondence Leoni Villano Bonamin, DVM, PhD,
Universidade Paulista, São Paulo, Brazil Research Center—UNIP, Graduation Program in Environmental and
2 IPEN—Institute of Energy and Nuclear Research, São Paulo, Brazil, Experimental Pathology, Universidade Paulista, Rua Dr Bacelar,
São Paulo, Brazil 1212—4th Floor, 04026-002 São Paulo, Brazil
3 DiagnOx Laboratory, Cherwell Innovation Centre, Upper Heyford, (e-mail: [email protected]; [email protected]).
Oxon, United Kingdom

Homeopathy 2021;110:244–255.

Abstract 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 ¼ 0.0018) and of chlorine/oxygen ratio (p < 0.0001) in suspended
Keywords micraggregates, suggesting changes in mercury bioavailability. A specific interaction of MC
► mercury 30cH with the solvatochromic dye ET33 (p ¼ 0.0017) was found.
► brine shrimp Conclusion Changes in hatching rate and possible changes in Hg bioavailability are
► homeopathy postulated as protective effects of MC 30cH on Artemia salina, by improving its
► ecotoxicology natural bioresilience processes.

received © 2021. The Faculty of Homeopathy. DOI https://doi.org/

November 16, 2020 All rights reserved. 10.1055/s-0041-1729562.
accepted after revision Georg Thieme Verlag KG, ISSN 1475-4916.
January 27, 2021 Rüdigerstraße 14,
published online 70469 Stuttgart, Germany
September 3, 2021
 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al. 245

Introduction are dependent on the dilution and agitation process of the

liquid (succussion), which is a crucial step in their manufac-
Mercury is a silvery metal, liquid at room temperature, ture.35–37 Since nanobubbles are present in these products in
and found naturally in the earth’s crust. It is traditionally large amounts,38 their role in the protective activity of
used in the manufacture of thermometers, electrodes, intoxications is under scrutiny. In addition, it is known
sphygmomanometers, barometers, and dye for paints. that the dilution and succussion processes also produce
Mercury oxide causes environmental contamination, in- specific changes in the electric properties of the solvent
volving water, plants, fish, birds and man, and its deposition related to initial dissolved substance, and these changes
in tissues is cumulative. The release of mercury into nature are observable by different techniques.39,40 It is possible
has several anthropogenic consequences.1–3 that the dilution and succussion process could modify the
Mercury can form organic mercury compounds (methyl- distribution of heavy metals between water and organisms,
mercury, ethylmercury, phenylmercury) or inorganic salts even in minute but measurable amounts.41,42
with chlorine, sulfur, or oxygen, accumulating in the envi- Solvatochromic dyes have been used as a sensitive method
ronment3,4 or remaining suspended in the air in particulate to identify succussed high dilutions.43–48 This method is
forms.5 Human contamination occurs mainly in the form of based on the capacity of these dyes to act as probes, revealing
methylmercury (organic form), through eating fish and changes in patterns of solvent/solute polarity in the presence
marine products, due to the tendency for bioaccumulation of homeopathic samples,43–46 through changes in their visi-
in these organisms.6,7 Ingestion can also occur in the inor- ble spectra. The method has been used since 2016 as a
ganic form.8 Poisoning by mercury chloride (HgCl2) occurs sensitive tool for evaluating homeopathic potencies.43 In
from waste of fluorescent lamps and ingestion of plants short, changes in dye absorption spectra reflect changes in
grown in contaminated soil.4,9 solvent/solute polarity after the addition of highly diluted
Following the mercury poisoning disaster in the 1950s at and agitated samples.43–46 The correspondence between
Minamata, Japan,1,9 efforts have been made to find solutions such reactivity and biological effects has been described in
to water decontamination and for mitigating the consequen- vitro.47 In a recent field study, the propagation of signals
ces of environmental pollution in exposed living beings.10 induced by homeopathic high potencies in large volumes of
Among several known remediation methods for removing water was detected by this technique.48
mercury from soil or water,3,11–15 a new technique has been In the current study, Artemia salina (brine shrimp) has
developed in recent years, using nano- and microbubbles to been used as an experimental model49–51 to test if Mercurius
facilitate the precipitation or fluctuation of contaminating corrosivus (MC), prepared from homeopathic manipulation
particles in water reservoirs.16 Nanobubbles have a diameter of HgCl2, can act as a remediation agent following sea-water
of 10 to 100 nm and can be stable for long periods, unlike contamination by a harmful concentration of the same salt.
macrobubbles that burst on the water surface. The pressure The proposed model was designed to observe any MC-
inside the nanobubbles is equivalent to four times the induced effects on nauplii hatching when challenged with a
atmospheric pressure; thus, when such bubbles burst, they concentration of HgCl2 able to kill 10% of the nauplii (Lethal
generate energy capable of producing free radicals of oxygen, Concentration 10%, or LC 10), this was chosen to mimic the
mainly OH, which are potential cation captors.17,18 Thus, real conditions of water contamination (►Supplementary
nanobubbles can also be used as a clean and sustainable tool Table S1, available online only). Besides the possibility of
of water purifying. The remediation of water contamination understanding some mechanisms related to an eventual
by mercury and methylmercury by nanobubbles has been protection, such putative effects could also be used as a
demonstrated recently.19,20 remediation tool to reduce the impact of water pollution in
In cases of acute mercury poisoning, chelating agents are living beings, considering a translational approach.
often used as antidote,2 but the treatment of chronic poison- Nauplii embryos tend to remain in the diapause stage
ing is still a challenge. The use of homeopathic preparations inside the cysts when in adverse conditions, including water
of the contaminating substance (isotherapeutics) is an estab- contamination by heavy metals. Only under favorable con-
lished treatment for intoxications caused by the same sub- ditions will nauplii cysts break their first lining after 20 hours
stance, being officially recognized in countries such as India and thereby hatch the embryos.52,53 The hatching phase
and Brazil.21,22 Experimental evidence arising from the involves singular physical and biochemical regulations to
method indicates recovery of compromised organic func- warrant the integrity of the embryo, such as sugars (treha-
tions by non-chelating mechanisms.23–29 Mercury isother- lose) able to produce a vitrification process of the shell,
apeutics have been observed as anti-genotoxic to mice making it very resistant to several aggressive stimuli. Heat
exposed to mutagenic substances, including several heavy shock proteins are produced in this phase too, protecting the
metals.30 Single cell cultures treated with homeopathic embryo’s life. A complete description of this process is given
Mercurius solubilis show higher production of ROS (reactive elsewhere.54 These features make Artemia salina a good
oxygen species), RNS (reactive nitrogen species), and several model to study bioresilience processes.
peptides relevant to inflammatory response.31 Bioremediation and bioresilience are related concepts.
There are reports showing that homeopathic products can The former defines a natural process used to treat or reduce
be important tools in organic and ecologically sustainable environmental chemical contamination, especially when
agriculture.32–34 The biological effects of such preparations using biological agents to promote it. By contrast,

Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
246 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al.

bioresilience is the capacity of any individual or species to urgent need of finding solutions to one of the world’s most
adapt to environmental changes. Thus, in this study, we serious contemporary concerns: water pollution.
postulate that isotherapeutic intervention could remediate
the effects of heavy metal exposition on living beings by Preparation of Homeopathic HgCl2 (Mercurius
means of the improvement of their own bioresilience Corrosivus)
mechanisms. Stock homeopathic potencies of MC were prepared from a
In addition, environmental variables important for the 1.0 M HgCl2 solution (SIGMA-MERCK, Saint Louis, United
physiology of marine organisms, including brine shrimps, States, CAS number 7487–94–7, prepared in HCl 1.0 M) at
such as time of exposure to HgCl2 and the phases of the a handling pharmacy accredited by ANVISA (Brazilian Na-
moon,55–57 were simultaneously studied, to aid in the tional Health Surveillance Agency) for this purpose. All
interpretation of results. Many marine species are suscep- preparations were made according to techniques described
tible to the lunar cycles and the consequent variations of in the official national guidelines.21,22
gravity, mainly in relation to reproductive functions and These stock potencies were prepared by serial centesimal
behavior.58 The swimming behavior of Artemia salina dilutions of the HgCl2 stock solution, with each step followed
changes according to the temperature at different levels by 100 vertical shakes (succussions) performed automatical-
of water column, which oscillate between the new moon ly in a mechanical arm (Denise—AUTIC, São Paulo, Brazil).
and the full moon, modifying their access to food and water Potencies were named according to the number of cycles
contaminants.59 In recent studies performed by our group, (dilutions/succussions). Thus, the 5, 29 and 199cH potencies
changes in the toxicity of sodium arsenate and lead chloride were supplied by the pharmacy and corresponded to the 5th,
on Artemia salina were observed according to different 29th and 199th centesimal dilutions, respectively, submitted
moon phases, with toxicity more evident during the full to succussion. All stock potencies were prepared in 70%
moon.54,60 alcohol, as recommended by ANVISA.21
Physicochemical analyses of water and investigation of
the interaction between MC and solvatochromic dyes were Preparation of Working Potencies
also performed to identify possible mechanisms involved in Working homeopathic potencies of HgCl2 (6, 30, 200cH) were
the effects of HgCl2 toxicity and their potential mitigation by prepared in the laboratory by 1:100 dilution of 5, 29 and
MC, as explained in the Discussion section. 199cH stock preparations, supplied by the pharmacy as
described above, followed by succussion. Working potencies
were prepared on the eve of the experiment, using as solvent
Material and Methods
sterile purified water obtained in a Direct-Q3 purification
Ethical Matters system with SmartPak Direct Q3 and Biopak filters (MERCK—
In the present study, vertebrate animals were not used. MILLIPORE, Darmstadt, Germany) and filtered through a
According to national (CONCEA) and international (EU 0.22 µm mesh filter (MILLIPORE, Burlington, United States).
Directive 2010/63/EU for animal experiments) legislation, After the final dilutions were ready, the bottles were closed
analysis of projects by an ethics committee is not neces- and subjected to automatic succussion in a mechanical arm
sary in cases where invertebrates (except cephalopods) are (Denise—AUTIC, São Paulo, Brazil), following the national
used. standards.21
Artemia salina has been used as a model for ecotoxicity Three types of controls were used. The first was made
studies since the 1950s. It is considered as an alternative method with purified sterile water submitted to succussion,
to replace the use of mammals or other vertebrate species, the second consisted solely of purified sterile water, with-
because of its sensorial characteristics typical of micro-crusta- out succussion, and the third was prepared from the
ceans of the phylum Brachiopoda. This species has an extremely centesimal homeopathic potency of the vehicle in sterile
low level of sentience, since its nervous system is very primitive, water, here named Ethilicum 1cH, containing 0.7% of alco-
being composed of a pair of nervous ganglion chains, linked to hol. The use of three different controls permitted the
one another by bundles of axons that control the vital functions identification of any effects associated with the vehicles
and movement. These animals respond to environmental stim- under serial agitation.
uli in an involuntary manner, with no emotional involvement All preparation and storage procedures and the handling
since there is no limbic system or similar structure in their of all medication were performed under sterile conditions in
nervous system. As in vertebrates similarly, nervous ganglions a laminar flow hood.
participate solely in autonomic functions.61
Herein, the objective was to verify, using an experimental Bottle Coding
model, if an isotherapeutic prepared from the mercury The bottles of MC working potencies and Ethilicum 1cH had
chloride itself could possibly mitigate the environmental their labels exchanged by a technician not involved in the
impact of exposure of living systems to toxic substances, experiment before the study began. The original labels and
such as heavy metals or pesticides. This model allows the respective codes were kept in a sealed envelope
extrapolation of the results to field conditions, with special throughout the test procedures and was opened only after
(but not exclusive) focus on marine biomes. This approach the end of the statistical analysis. Thus, all tests were
might yield clear environmental benefit, responding to the performed blind.

Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al. 247

Hatching of Artemia Salina Cysts The plates containing the cysts were placed in a hand-
The experimental manipulation for hatching of nauplii fol- made Faraday cage and illuminated 24 hours a day with 50-
lowed these steps: 60 Hz LED light (IT-666, Yi Teng, China) to promote hatching
1. Preparation of artificial sea water in a stable environment. They remained open to allow
One liter of artificial sea water was prepared by adding 30 g enough oxygenation of water surface, necessary to promote
of artificial sea salt (Ocean Fish—PRODAC, Citadella PD, Italy) in hatching. The laboratory environmental conditions were
1.0 L of distilled water, under constant agitation. Then, food monitored throughout the experimental period. The room
was included in a proportion of 5.0%. The food consisted of a temperature average was 22.83  0.43°C and the electromag-
suspension of powdered biological yeast (Saccharomyces cer- netic flow near the plates was 0.07  0.06 µT at 50Hz (Smart-
evisiae) (5 mg/mL), previously prepared in artificial seawater. Sensor Intel Instruments, AS 1392, Singapore).
2. Definition of the experimental groups Moon phase was also observed during the whole experi-
Nine 96-well plates were used weekly, within four weeks mental period and any statistical correlations between moon
of experiment. Thus, 36 plates were used throughout the phases and hatching rates were noted. All moon phases were
study. For each plate, treatments were dispensed in rows of included within this study period.
12 wells, each containing cysts in the process of hatching, After recordings, made at 24 and 48 hours of incubation,
according to the following design: the plates were frozen, and the contents stored for later
physicochemical analysis.
• Cysts not challenged with HgCl2 and untreated.
• Cysts challenged with HgCl2 and treated with pure sterile
water. Physicochemical Analysis of Water
• Cysts challenged with HgCl2 and treated with succussed
Scanning Electron Microscopy and X-Ray Dispersive
pure sterile water.
Energy Spectroscopy (JEOL 6510)
• Cysts challenged with HgCl2 and treated with Ethilicum
The contents of wells were collected from randomly select-
1cH.
ed plates and grouped into sample pools for each type of
• Cysts challenged with HgCl2 and treated with MC 6cH.
treatment (controls or MC 6cH, MC 30cH, MC 200cH), in a
• Cysts challenged with HgCl2 and treated with MC 30cH.
manner such that each treatment was evaluated in tripli-
• Cysts challenged with HgCl2 and treated with MC 200cH.
cate. Each pool was centrifuged at 10,000 RPM (11,068 g,
The cysts (Global Biotérios, São Paulo, Brazil) were kept Centrifuge MCA mod.1–4K, Sigma, Osterode am Harz,
dry (290,000 units per gram) in an airtight desiccator with Germany) for 60 minutes, to separate the biological sedi-
reduced humidity, containing a layer of silica gel at the ment from water. Then, the supernatant was filtered
bottom. Immediately before use, the cysts were homo- through a 0.22 µm mesh filter (Millipore, Burlington, United
geneously suspended in artificial seawater (75 mg of cysts States) and centrifuged again at 10,000 RPM (11,068 g) for
per 200 mL) to obtain six to eigth cysts per 100 µL. This was 1 hour, to obtain non-biological particulate sediment and
the volume inserted in each well. the respective supernatant, which was stored for measure-
3. Challenging of cysts with mercury chloride ment of soluble mercury. At the end of this process, two
Mercury chloride was inserted into the water at the same kinds of sediment (biological and inorganic) were used in
time as the cysts at a concentration equivalent to LC 10 scanning electron microscopy/X-ray dispersive energy spec-
(lethal concentration 10%) or 5.0 µg/mL (containing 370 µM troscopy (SEM/EDS) analysis.
of total mercury—THg—per well), as previously established The analysis of inorganic sediment was performed using
in a pilot experiment. A low (but harmful) concentration of round basement coverslips cleaned with acetone and dried.
mercury (LC 10) was chosen for this experiment to mimic They were previously metallized in a vacuum chamber
usual conditions of mild environmental contamination as- (Desk V, Denton Vacuum, Moorestown, New Jersey, United
sociated with processes of chronic toxicity.7,62 Aliquots of MC States) with a gold foil, and 10 µL of each sediment removed
6, 30 or 200cH were inserted in each well at the time of from the bottom, just after centrifugation, was placed on the
intoxication, at a volume equal to 10% of the final volume of coverslip surface as droplets, to avoid the liquid spreading.
sea water. Thus, the final concentration of residual alcohol in The coverslips were then placed in an oven at 50°C until the
each well was 0.07%, not enough to promote biological effects droplets dried. The analyses were made in a scanning elec-
on the brine shrimps.63 tron microscope (JSM 6510, JEOL, Tokyo, Japan) with the EDS
4. Analysis of hatching rate and nauplii viability system by a primary beam of accelerated electrons set at
After 24 and 48 hours, each well was observed using a digital 15kV.
pen microscope (Digital Microscope with Zoom 1000x R Camera For the analysis of the biological sediment, the samples
2.0 Mega Pixels USB 6 Leds, Beijing, China), and data were were fixed in 3% buffered formaldehyde for 24 hours at 4°C,
recorded (AMCAP, Beijing, China) on a computer (Yoga520, washed in distilled water, and dehydrated in an increasing
Lenovo, Brazil) for counting of hatched/non-hatched cysts. alcoholic graduation bath, starting from 30%, for 5 minutes at
5. Experimental design and environment control each graduation until finally reaching three baths in ethanol
Plating was performed on three consecutive days (three 100%. Then they were taken to the critical point in HMDS
series in triplicates) in a week, for 4 weeks, totaling 36 (hexamethyldisilazane, Sigma-Merck, United States) with
repetitions and 14,468 hatched nauplii for analyses. three immersions of 5 minutes. After total drying, the

Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
248 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al.

samples were placed in round glass coverslips (22 mm) The LD detection limit obtained was 0.4 μg/L; the preci-
adhered to each stub. The metallization of biological samples sion and accuracy of the measurement was confirmed by the
was performed in a vacuum chamber (as described above), recovery of 90% of the total mercury concentration with a
using a gold foil. The images were captured by SEM at 1000x certified reference material, DORM-4, as described by Pecor-
magnification and five random fields per sample were aro et al.42 The high accuracy of this method allows repro-
recorded. EDS was used to identify the chemical elements ducibility and reliability of analysis even in small samples.
present. Silica and gold were not considered for evaluation to One measurement was initially made for each sample.
avoid bias, as these same elements were part of the metal- Those samples presenting discrepant values in relation to the
lized coverslip structure. median (12.45 ppm) were selected to be measured in differ-
Similarly, samples of biological sediment deposited on ent replications, to check their validity.
previously metallized coverslips were analyzed by area. The
droplet area delimited for analysis comprised a 2.5 mm  3.5 Interaction of MC Potencies with Solvatochromic Dyes
mm square, covering at least 80% of the material surface. In Samples of MC working potencies were submitted to spec-
this case, samples were analyzed at 30x magnification, using trophotometry using different solvatochromic dyes. Dyes
the same equipment and conditions described were chosen to cover all the visible spectra, in order to
above. ►Fig. 1A and 1B show typical SEM images of non- find the most sensitive of MC potencies (►Supplementary
biological and biological sediments, respectively. Table S2, available online only). The dyes were diluted in
absolute alcohol on the evening prior to use and stored in
Determination of Total Mercury (THg) by Thermal sterile amber glass flasks at room temperature, and isolated
Decomposition, Amalgamation and Atomic Absorption from any strong electromagnetic fields.
using a Direct Hg Analyzer Manipulation of dyes and cuvettes was performed in a
The measurement of soluble mercury was performed in a DMA- laminar flux hood. Samples and dye solutions were filtered in
80 Hg analyzer (Milestone, Sorisole, Italy). The mercury content a 0.22 µm mesh filter (Millipore, Burlington, United States) just
was determined based on thermal decomposition, amalgam- before use. The ratio between dye solution and sample in the
ation and atomic absorption.41 A small amount of filtered water cuvettes was 60:1. The internal controls consisted of succussed
sample (20–200 μL) was placed in each quartz boat, which was or non-succussed sterile purified water. Readings were per-
then introduced into an oven enriched with oxygen, and heated formed using a UV-vis spectrophotometer (FEMTO 800 XI, São
to 750°C, to proceed the thermal decomposition. Paulo, Brazil). The absorbance of each dye solution was scanned
In this method, halogens, nitrogen oxides and sulfur between 350 and 800 nm, with a resolution of 1 nm, to find the
released in decomposition are retained in a catalyst. Mercury exact wavelength corresponding to the peak of absorbance.
is selectively amalgamated in an amalgamation cell, where Analyses were performed at the wavelength corresponding to
mercury is separated from any decomposition products. this peak. All analyses were done in triplicate. Purified water
Finally, the amalgamator is heated quickly, releasing the was used as the absorbance baseline, to calibrate the equipment
mercury vapor to be quantified by atomic absorption. immediately before each measurement series.

Fig. 1 (A) Droplet generated from water sample treated with Mercurius corrosivus 30cH, enlarged 30x and deposited on a previously metallized
coverslip; (B) Captured area of biological sediment obtained from water treated with Mercurius corrosivus 30cH, in 1000x magnification. The
smaller spheres correspond to the yeasts used to feed nauplii, the oval structure represents an Artemia salina cyst. The analysis was performed
using scanning electron microscopy (SEM) and X-ray dispersive energy spectroscopy (EDS) (JEOL 6510). The marked straight margins correspond
to the analyzed area.

Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al. 249

All experimental procedures involving physicochemical

analysis were also performed blind, with sample and control
codes only revealed following the statistical analyses.

Statistical Analysis
Two-way analysis of variance (ANOVA) with mixed models
was used for evaluating the effect of different treatments and
the simultaneous influence of the moon phases on the cysts
hatching rate, at both observation times (24 and 48 hours).
The relative hatch rate was defined as:
Hatching rate (%) ¼ nauplii/non-hatched cysts.
The effect size was also observed using partial eta-
squared (ɳ2) calculation, and Cohen’s criteria were used to
define the test power. The experimental unit was the sum of
specimens observed in all of 12 wells from each plate: thus,
N ¼ 9 per group.
The analysis of the sedimented elements was done by
two-way ANOVA (for treatment and biological replicate) and
the analyses of the solvatochromic dyes assays were done by
Fig. 2 Effect of treatment on hatching rate. The values represent
one-way ANOVA, followed by Tukey’s test.
mean  95% confidence interval of the whole population, taken from
In all cases, the assumptions of the models were all data together, independent of the moon phase and time of
investigated, respecting Normality and homogeneity. observation. A significant main effect of the treatment was observed
When necessary, outliers were removed, using the Tukey on the relative hatching rate of cysts, according to two-way ANOVA
criterion. In this case, the rule was applied if the values with mixed models (F (6; 224) ¼ 10.260, p < 0.001, partial ɳ 2 ¼ 0.216).
Two levels of significance were seen after Tukey post-test: (a) signif-
exceeded limits of 1.5 times the interquartile range of the
icant difference between the unchallenged group versus all chal-
group above the third quartile, or 1.5 times the inter- lenged groups which were exposed to HgCl2 at LC10 (p < 0.05); (b)
quartile range of the group below the first quartile. The significant difference (#) between the treatment with Mercurius
level of significance α was set at 5%: thus the effects that corrosivus (MC) 30cH versus unchallenged (p < 0.001), water
obtained a chance of error p < 0.05 were considered as (p ¼ 0.018) and succussed water (p ¼ 0.012), showing its specific
effect. ANOVA, analysis of variance.
significant.

Results
Analysis of Cyst Hatching
The main effect of treatment with controls or MC samples for
HgCl2 challenged nauplii is shown in ►Fig. 2, in which all 36
repetitions were considered, taking all data together, inde-
pendent of the observation time or the moon phase. Expo-
sure of Artemia salina to HgCl2 at LC 10 induced a significant
decrease in hatching rate (p < 0.05), remembering that cysts
are forms of resistance of the species and reduction in
hatching rate is expected in hostile environments. However,
incubation of exposed cysts with MC 30cH resulted in an
additional and significant reduction of the cysts hatching
rate compared with water and succussed water controls
(p ¼ 0.018 and p ¼ 0.012, respectively; ɳ2¼ 0.216). It there-
fore appeared to be a specific effect of this treatment,
considering the whole population behavior exposed to this
challenge.
The observation of a statistical correlation between moon
phase and hatching rate was important to separate any lunar
effect from the effects of MC potencies. Results in ►Fig. 3 Fig. 3 Effect of the phases of the moon on hatching. The values
show a significant effect of the full and waning moon phases represent mean  95% confidence interval for the mean. A significant
on the rate of hatching of the cysts. During the crescent main effect of the moon phase was observed on the relative hatching
rate of cysts, according to two-way ANOVA with mixed models
moon, the unchallenged cysts mostly hatched after 48 hours.
(F (3; 224) ¼ 49.699, p < 0.001, partial ɳ 2 ¼ 0.400). Tukey’s post-test
At full moon, on the contrary, approximately 80% of the cysts indicated significant difference between full, waning, or new moon
had already hatched within 24 hours. However, no statistical versus crescent moon (p < 0.001) and new moon versus waning moon
interaction between the moon phases and the treatments (#p < 0.001). ANOVA, analysis of variance.

Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
250 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al.

was seen (F (18; 224) ¼ 1.605, p ¼ 0.060; ɳ2 ¼ 0.400). In short,

the natural oscillations of hatching rate relative to moon
phase were independent of the effect of MC 30cH.

Analysis of Insoluble Mercury and Particulate

Elements
There was no measurable precipitation of mercury in the
analyzed biological sediments and only carbon and oxygen
were above the detection limit. This is to be expected since
they are the main constituents of organic material. In the
mineral sediments, traces of mercury (less than 1%) were
found only in one of the samples of Ethilicum 1cH and
succussed water. Aluminum, boron, bromine, barium, potas-
sium, niobium and sulfur were excluded from the analysis
because they were present in less than 2% of the general
average. Among the other measurable elements (oxygen,
sodium, magnesium, chlorine and calcium) found in these
samples, only chlorine and oxygen warranted further inves-
tigation, being found in higher percentage in samples Fig. 5 Percentage of oxygen in mineral sediments identified by EDS
obtained from water treated with MC 30cH and MC 200cH. from samples of filtered water obtained from the cyst cultures. The
Curiously, the oxygen percentage was lower in the mineral analyses were processed in biological independent triplicates. The
total volume of each replicate was deposited on the metalized
sediments taken from water treated with MC 200cH, in an
coverslip and left to dry. The values represent mean  95% confidence
inverse relation to that for chlorine (►Figs. 4 and 5). The interval (for treatments—circles). A significant main effect of the
whole profile of the EDS spectra obtained from all samples of treatment was observed on the percentage of oxygen, according to
mineral sediments is shown in ►Supplementary Fig. S1 two-way ANOVA (F (6; 14) ¼ 11.39, p < 0.001, partial ɳ 2 ¼ 0.830).
(available online only). Tukey’s post-test indicated significant difference between MC 200cH
versus all other conditions (#p < 0.02) and MC 30cH versus unchal-
lenged (p ¼ 0.033). EDS, X-ray dispersive energy spectroscopy.

Analysis of Soluble Mercury Concentration Using Hg

DMA-80 Analyzer (Milestone)
The concentration of total soluble mercury (THg) found in
water was three times higher in the group treated with MC
30cH, compared with the other experimental conditions
(►Fig. 6).

Fig. 4 Percentage of chlorine in mineral sediments, identified by EDS

from samples of filtered water obtained from the cyst cultures. The
analyses were processed in biological independent triplicates. The
total volume of a replicate was applied to the metalized coverslip and
left to dry. The values represent mean (for biological triplicates—bars)
and mean  95% confidence interval (for treatments—circles). A sig-
nificant main effect of the treatment was observed for the percentage
of chlorine, according to two-way ANOVA (F(6; 14) ¼ 53.978, p < 0.001,
partial ɳ 2 ¼ 0.959); Tukey’s post-test indicated significant difference Fig. 6 Concentration of total soluble mercury (THg)—μg/L
between MC 30cH or MC 200cH versus unchallenged (p < 0.001), and (F (6,7) ¼ 12.80, p ¼ 0.0018, partial ɳ 2 ¼ 0.916); p  0.05 in relation to
MC 6cH, 30cH, or 200cH versus water (#p < 0.02). EDS, X-ray disper- the other conditions, according to Tukey’s post-test. Each point
sive energy spectroscopy. represents an individual value.

Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al. 251

environmental stressful stimuli, in which there is arrest of

embryo development at the gastrula stage, keeping a low
metabolic level. These events are controlled by the expres-
sion of specific genes, mainly those related to the composi-
tion of the cyst wall, allowing the deposition of trehalose, a
disaccharide that cannot be decomposed to glucose. This
sugar forms H bonds with proteins and phospholipids,
forming a kind of bioglass, in a process called vitrification,
allowing the maintenance of internal stability.54,60,67–73
Besides this first adaptative level of hatching reduction,
another deeper level of hatching inhibition was revealed
after the analysis of the whole population, independent of
time and moon phase, with high statistical significance and
large effect size against the controls, as observed after
treatment with MC 30cH (►Fig. 2). Thus, this effect was
considered as specific, and able to improve the natural
adaptative process of Artemia salina in the face of a harmful
environment. Results were obtained from the dataset of 36
repetitions, adding 14,468 nauplii, which reflects a consis-
Fig. 7 Absorbance of the dye ET33 using visible light spectropho-
tometry after adding different MC dilutions and controls. tent population effect.
(F (5,10) ¼ 9.137, p ¼ 0.0017, partial ɳ 2 ¼ 0.820); p ¼ 0.04 in relation to The physicochemical analyses revealed an increase of
the other groups, according to Tukey’s post-test. soluble THg in samples treated with MC 30cH, and interac-
tivity of MC 30cH itself to a specific solvatochromic dye (ET
Interaction of Homeopathic Samples with 33) was also observed (►Fig. 7). Solvatochromic dyes are
Solvatochromic Dyes very sensitive to the presence of homeopathic preparations,
The interaction of homeopathic potencies with solvatochro- producing subtle but precise changes in the peak of absor-
mic dyes was selective: only MC 30cH showed interaction bance,43–47 and different patterns of reactivity may corre-
with ET 33, by reducing absorbance in relation to the controls spond to different biological activities47 through changes in
(►Fig. 7). The other dyes did not show any interaction with the polarity of the medium (solutes and/or water). According
MC dilutions. to recent studies performed on solvatochromic dyes, changes
in optical density can occur even when dye molecules are
encapsuled in β-cyclodextrin, avoiding any molecular con-
Discussion
tact of dyes with water structures and aggregates, suggesting
The results obtained in this study show the effects of that these dyes, in the presence of homeopathic potencies,
various preparations of MC in modulating the toxicity of are more electrochromic than solvatochromic.46 Thus,
HgCl2 LC 10 (lethal concentration of 10%) exposure in effects related to mere biochemical interactions between
relation to hatching of Artemia salina nauplii. The effect additional ions present in water bulk and these dyes are
of MC 30cH was the most significant and appeared to be unlikely.
independent of external variables such as the moon phases Hypothetically, the MC 30cH effects on hatching, THg
and the time of observation. soluble concentration and ET33 dye spectra could be associ-
Exposure to HgCl2 reduced the hatch rate, indicating a ated with a common biophysical mechanism, possibly relat-
defense process of the cysts. This non-specific effect can be ed to a particular excitation state of the liquid medium.
hypothetically attributed to oxidative stress on the em- The results allow for the use of these homeopathic
bryo,64 due to the presence of nano- and air microbubbles potencies as agents in a simple and low-cost remediation
(oxygen) and/or the presence of hydroxyl radicals generated procedure, able to improve natural bioresilience processes
during the succussion process of a polar medium.17,18,38 and be monitored by solvatochromic dyes, even in a large
Cysts are forms of resistance of the species and their perma- volume of water, as previously reported.48
nence as such reveals a well-known resilience mechanism, 1Circalunar variations are known to be important modula-
involving the production of chaperone (p26, ArHsp21, tion factors on the physiological processes of several marine
ArHsp22, and artemin) and other proteins by the em- species. In 2020, Andreatta and Tessmar-Raible pointed out
bryo.65–67 Similarly, the treatment with Ethilicum 1cH (suc- connections between rhythms of metabolic and endocrine
cussed 0.7% alcoholic solution) was also related to a first level pathways and circalunar variations,57 making it possible to
of cyst protection. According to Sandri et al, potentized identify molecular markers, such as gonadotrophin-releasing
alcohol cannot be considered as an inert vehicle, but a hormone and neuropeptides, related to the expression of
specific homeopathic medicine, thus a comparative “clock genes” in several marine species.73 Lunar cycles also
substance.68 promote oscillation and synchronization of other biological
This is part of an adaptation process used by Artemia spp., processes, such as general activity, photosensitivity, and verti-
called “diapause”, to preserve the species when exposed to cal migration of zooplankton.55,57,73 Environmental variables,

Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
252 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al.

such as the moon phase, also seem to be involved in Artemia homeopathic potencies of mercury against heavy metal
salina biological processes, as shown herein, but they were not poisoning is also reported in rodents and macrophages
able to change the specific effects of MC 30cH. in vitro.30,31 These results suggest that the use of isother-
Artemia salina seemed to be a good study model for the apeutic preparations to mitigate the effects of water pollu-
formulation of strategies to mitigate the impact of water tion on living systems could be a plausible tool in an eventual
contamination on local living systems,70 by improving their environmental management program and be proposed as a
resilience to harmful stimuli. MC 30cH seems to optimize new approach for sustainable and low-cost health promotion
this natural resilience, suggesting it as a potential adjuvant for aquatic organisms32,33 that are exposed to low, but
tool to reduce the environmental impact of residual levels of constant, concentrations of mercury.81,86 Other studies per-
mercury, especially if applied in association with traditional formed by our group also point to similar effects, using the
processes of clearance of heavy metals from water.10–15 This same methods to study other toxins.87,88
conclusion is in accordance with previous studies on homeo-
pathic potencies that often show a constant pattern of action:
Conclusion
the facilitation of adaptive processes of the organism against
aggressive agents in a non-linear way,74–80 involving the The exposure of Artemia salina cysts to HgCl2 inserted in
modulation of immunological and metabolic processes, as seawater in a lethal concentration of 10% (LC10) leads to
well as gene expression.27–35 The findings described here delay of hatching—a natural adaptative process of the species
follow these same pro-adaptative features. to survive in harmful environments. The addition of MC 30cH
Another aspect that deserves attention is the property of into seawater led to a significant enhancement of this
nanobubbles to present electrically charged interfaces, adsorb- response, independently of external variables.
ing suspended solids in seawater, which could suggest that the The THg distribution among living and non-living com-
simple insertion of water subjected to succussion into seawater partments in the aquatic medium and the interactivity of MC
could trigger some non-specific purification process.38,81–83 30cH with ET33 dye can each help to build hypotheses to
Moreover, the oxygen contained in nanobubbles reduces the explain this peculiar biological effect.
synthesis of methylmercury in water.19,20 The study presents the potential use of isotherapy as a
An alternative explanatory hypothesis for the effects of sustainable tool for bioresilience management in cases of
MC 30cH would be the stock of oxygen and chlorine in such water contamination.
deposits, able to be released into water by diffusion. Chlorine
Highlights
could be a source of mercury stabilization in water, in the
form of HgCl2 or CH3HgCl,82 preventing mercury’s absorp- • Mercurius corrosivus (MC) was investigated as a puta-
tion by the exposed nauplii and reducing its toxicity. This tive bioremediatory agent.
hypothesis is reinforced by the fact that MC 30cH was the • Cysts of Artemia salina exposed to low concentrations of
only treatment related to higher concentration of soluble HgCl2 were used as experimental model.
THg in water, compared with the other treatments. • The dilution MC 30cH potentiated the delay of cysts
In all cases, after 48 hours, when almost 100% of cysts were hatching, an adaptative process.
hatched, the level of THg in water was 200 times lower than the • It changed the balance of soluble THg, chlorine and
content inserted into the wells. A certain degree of absorption oxygen contents in water.
of HgCl2 by yeasts (used as food for nauplii) associated with the • It seems to change medium polarity, as indicated by its
natural volatilization of HgCl214,83,84 could be the reason of interaction with solvatochromic dyes.
this decay, since there was no detectable deposition of mercu- • MC 30cH is proposed as a potential tool for improving
ry in organic and inorganic microaggregates. bioresilience to mercury.
By contrast, reduction in oxygen in the inorganic sediment
was associated with an increase of chlorine in the samples
treated with MC 200cH, suggesting possible anionic exchanges
Supplementary Files
by the nauplii. These animals can perform “osmoregulation”
using the “neck organ”, a Cl-excretory gland, which gives to Supplementary Table S1 Pilot experiment to determine
Artemia salina the ability to adapt very well to hyper-saline and the correspondence between HgCl2 concentrations and
harsh environments. This gland works by means of the enzyme 48-hour-old nauplii lethality
Naþ/Kþ-ATPase, whose activity depends on aerobic metabo- Supplementary Table S2 Standardized dyes used in this
lism.52 The suggested hypothesis to explain the effects of the experiment
MC 200cH, in this case, would be an increase in the activity of Supplementary Fig. S1 Profile of the spectrum of EDS
the “neck organ”, by mechanisms still unknown. analyses, from mineral sediment obtained by centrifu-
The protective activity of MC against intoxications is gation of filtered sea water.
described in previous studies. Jäger et al, in 2019, observed
protective effects of specific homeopathic potencies of mer-
Conflict of Interest
cury chloride in Lemna gibba L. intoxicated by the same
substance.85 Likewise, the protective action of specific None declared.

Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al. 253

Acknowledgements 15 Wang J, Shaheen SM, Anderson CWN, et al. Nanoactivated carbon

We are grateful to the Pharmacy HN Cristiano, São Paulo, reduces mercury mobility and uptake by Oryza sativa L: mecha-
and Dr. Amarylis Toledo César, the responsible Pharmacist, nistic investigation using spectroscopic and microscopic techni-
ques. Environ Sci Technol 2020;54:2698–2706
for manufacturing the study’s Mercurius corrosivus, which
16 Xiao W, Ke S, Quan N, et al. The role of nanobubbles in the
was prepared from the salt purchased from Sigma-Merck. precipitation and recovery of organic-phosphine-containing
We thank the University Paulista (UNIP) for the scholar- beneficiation wastewater. Langmuir 2018;34:6217–6224
ships of Andreia A.G. Pinto, Michelle S. Aguiar and Sandra A. 17 Agarwal A, Ng WJ, Liu Y. Principle and applications of microbubble
G. Pinto, and CAPES-Prosup for the scholarship of Ednar do and nanobubble technology for water treatment. Chemosphere
2011;84:1175–1180
Nascimento Coimbra, Mirian Y.D. Nagai and Suham Now-
18 Takahashi M, Chiba K, Li P. Free-radical generation from collapsing
rooz. Steven Cartwright gratefully acknowledges funding
microbubbles in the absence of a dynamic stimulus. J Phys Chem B
from Standard Homeopathic Company/Hylands, United 2007;111:1343–1347
States. We also thank Wilton Pereira dos Santos for techni- 19 Ji X, Liu C, Pan G. Interfacial oxygen nanobubbles reduce methyl-
cal support in the aquatic organism laboratory. mercury production ability of sediments in eutrophic waters.
Ecotoxicol Environ Saf 2020;188:109888
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