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

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
Introduction
Mercury is a silvery metal, liquid at room temperature,
and found naturally in the earth’s crust. It is traditionally
used in the manufacture of thermometers, electrodes,
sphygmomanometers, barometers, and dye for paints.
Mercury oxide causes environmental contamination, in-
volving water, plants, fish, birds and man, and its deposition
in tissues is cumulative. The release of mercury into nature
has several anthropogenic consequences.1–3
Mercury can form organic mercury compounds (methyl-
mercury, ethylmercury, phenylmercury) or inorganic salts
with chlorine, sulfur, or oxygen, accumulating in the envi-
ronment3,4 or remaining suspended in the air in particulate
forms.5 Human contamination occurs mainly in the form of
methylmercury (organic form), through eating fish and
marine products, due to the tendency for bioaccumulation
in these organisms.6,7 Ingestion can also occur in the inor-
ganic form.8 Poisoning by mercury chloride (HgCl2) occurs
from waste of fluorescent lamps and ingestion of plants
grown in contaminated soil.4,9
Following the mercury poisoning disaster in the 1950s at
Minamata, Japan,1,9 efforts have been made to find solutions
to water decontamination and for mitigating the consequen-
ces of environmental pollution in exposed living beings.10
Among several known remediation methods for removing
mercury from soil or water,3,11–15 a new technique has been
developed in recent years, using nano- and microbubbles to
facilitate the precipitation or fluctuation of contaminating
particles in water reservoirs.16 Nanobubbles have a diameter
of 10 to 100 nm and can be stable for long periods, unlike
macrobubbles that burst on the water surface. The pressure
inside the nanobubbles is equivalent to four times the
atmospheric pressure; thus, when such bubbles burst, they
generate energy capable of producing free radicals of oxygen,
mainly OH, which are potential cation captors.17,18 Thus,
nanobubbles can also be used as a clean and sustainable tool
of water purifying. The remediation of water contamination
by mercury and methylmercury by nanobubbles has been
demonstrated recently.19,20
In cases of acute mercury poisoning, chelating agents are
often used as antidote,2 but the treatment of chronic poison-
ing is still a challenge. The use of homeopathic preparations
of the contaminating substance (isotherapeutics) is an estab-
lished treatment for intoxications caused by the same sub-
stance, being officially recognized in countries such as India
and Brazil.21,22 Experimental evidence arising from the
method indicates recovery of compromised organic func-
tions by non-chelating mechanisms.23–29 Mercury isother-
apeutics have been observed as anti-genotoxic to mice
exposed to mutagenic substances, including several heavy
metals.30 Single cell cultures treated with homeopathic
Mercurius solubilis show higher production of ROS (reactive
oxygen species), RNS (reactive nitrogen species), and several
peptides relevant to inflammatory response.31
There are reports showing that homeopathic products can
be important tools in organic and ecologically sustainable
agriculture.32–34 The biological effects of such preparations
Homeopathy
Pinto et al. 245
are dependent on the dilution and agitation process of the
liquid (succussion), which is a crucial step in their manufac-
ture.35–37 Since nanobubbles are present in these products in
large amounts,38 their role in the protective activity of
intoxications is under scrutiny. In addition, it is known
that the dilution and succussion processes also produce
specific changes in the electric properties of the solvent
related to initial dissolved substance, and these changes
are observable by different techniques.39,40 It is possible
that the dilution and succussion process could modify the
distribution of heavy metals between water and organisms,
even in minute but measurable amounts.41,42
Solvatochromic dyes have been used as a sensitive method
to identify succussed high dilutions.43–48 This method is
based on the capacity of these dyes to act as probes, revealing
changes in patterns of solvent/solute polarity in the presence
of homeopathic samples,43–46 through changes in their visi-
ble spectra. The method has been used since 2016 as a
sensitive tool for evaluating homeopathic potencies.43 In
short, changes in dye absorption spectra reflect changes in
solvent/solute polarity after the addition of highly diluted
and agitated samples.43–46 The correspondence between
such reactivity and biological effects has been described in
vitro.47 In a recent field study, the propagation of signals
induced by homeopathic high potencies in large volumes of
water was detected by this technique.48
In the current study, Artemia salina (brine shrimp) has
been used as an experimental model49–51 to test if Mercurius
corrosivus (MC), prepared from homeopathic manipulation
of HgCl2, can act as a remediation agent following sea-water
contamination by a harmful concentration of the same salt.
The proposed model was designed to observe any MC-
induced effects on nauplii hatching when challenged with a
concentration of HgCl2 able to kill 10% of the nauplii (Lethal
Concentration 10%, or LC 10), this was chosen to mimic the
real conditions of water contamination (►Supplementary
Table S1, available online only). Besides the possibility of
understanding some mechanisms related to an eventual
protection, such putative effects could also be used as a
remediation tool to reduce the impact of water pollution in
living beings, considering a translational approach.
Nauplii embryos tend to remain in the diapause stage
inside the cysts when in adverse conditions, including water
contamination by heavy metals. Only under favorable con-
ditions will nauplii cysts break their first lining after 20 hours
and thereby hatch the embryos.52,53 The hatching phase
involves singular physical and biochemical regulations to
warrant the integrity of the embryo, such as sugars (treha-
lose) able to produce a vitrification process of the shell,
making it very resistant to several aggressive stimuli. Heat
shock proteins are produced in this phase too, protecting the
embryo’s life. A complete description of this process is given
elsewhere.54 These features make Artemia salina a good
model to study bioresilience processes.
Bioremediation and bioresilience are related concepts.
The former defines a natural process used to treat or reduce
environmental chemical contamination, especially when
using biological agents to promote it. By contrast,
Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
246 Mercurius Corrosivus and Bioresilience of Artemia Salina
bioresilience is the capacity of any individual or species to
adapt to environmental changes. Thus, in this study, we
postulate that isotherapeutic intervention could remediate
the effects of heavy metal exposition on living beings by
means of the improvement of their own bioresilience
mechanisms.
In addition, environmental variables important for the
physiology of marine organisms, including brine shrimps,
such as time of exposure to HgCl2 and the phases of the
moon,55–57 were simultaneously studied, to aid in the
interpretation of results. Many marine species are suscep-
tible to the lunar cycles and the consequent variations of
gravity, mainly in relation to reproductive functions and
behavior.58 The swimming behavior of Artemia salina
changes according to the temperature at different levels
of water column, which oscillate between the new moon
and the full moon, modifying their access to food and water
contaminants.59 In recent studies performed by our group,
changes in the toxicity of sodium arsenate and lead chloride
on Artemia salina were observed according to different
moon phases, with toxicity more evident during the full
moon.54,60
Physicochemical analyses of water and investigation of
the interaction between MC and solvatochromic dyes were
also performed to identify possible mechanisms involved in
the effects of HgCl2 toxicity and their potential mitigation by
MC, as explained in the Discussion section.
Material and Methods
Ethical Matters
In the present study, vertebrate animals were not used.
According to national (CONCEA) and international (EU
Directive 2010/63/EU for animal experiments) legislation,
analysis of projects by an ethics committee is not neces-
sary in cases where invertebrates (except cephalopods) are
used.
Artemia salina has been used as a model for ecotoxicity
studies since the 1950s. It is considered as an alternative method
to replace the use of mammals or other vertebrate species,
because of its sensorial characteristics typical of micro-crusta-
ceans of the phylum Brachiopoda. This species has an extremely
low level of sentience, since its nervous system is very primitive,
being composed of a pair of nervous ganglion chains, linked to
one another by bundles of axons that control the vital functions
and movement. These animals respond to environmental stim-
uli in an involuntary manner, with no emotional involvement
since there is no limbic system or similar structure in their
nervous system. As in vertebrates similarly, nervous ganglions
participate solely in autonomic functions.61
Herein, the objective was to verify, using an experimental
model, if an isotherapeutic prepared from the mercury
chloride itself could possibly mitigate the environmental
impact of exposure of living systems to toxic substances,
such as heavy metals or pesticides. This model allows
extrapolation of the results to field conditions, with special
(but not exclusive) focus on marine biomes. This approach
might yield clear environmental benefit, responding to the
Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
Pinto et al.
urgent need of finding solutions to one of the world’s most
serious contemporary concerns: water pollution.
Preparation of Homeopathic HgCl2 (Mercurius
Corrosivus)
Stock homeopathic potencies of MC were prepared from a
1.0 M HgCl2 solution (SIGMA-MERCK, Saint Louis, United
States, CAS number 7487–94–7, prepared in HCl 1.0 M) at
a handling pharmacy accredited by ANVISA (Brazilian Na-
tional Health Surveillance Agency) for this purpose. All
preparations were made according to techniques described
in the official national guidelines.21,22
These stock potencies were prepared by serial centesimal
dilutions of the HgCl2 stock solution, with each step followed
by 100 vertical shakes (succussions) performed automatical-
ly in a mechanical arm (Denise—AUTIC, São Paulo, Brazil).
Potencies were named according to the number of cycles
(dilutions/succussions). Thus, the 5, 29 and 199cH potencies
were supplied by the pharmacy and corresponded to the 5th,
29th and 199th centesimal dilutions, respectively, submitted
to succussion. All stock potencies were prepared in 70%
alcohol, as recommended by ANVISA.21
Preparation of Working Potencies
Working homeopathic potencies of HgCl2 (6, 30, 200cH) were
prepared in the laboratory by 1:100 dilution of 5, 29 and
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
sterile purified water obtained in a Direct-Q3 purification
system with SmartPak Direct Q3 and Biopak filters (MERCK—
MILLIPORE, Darmstadt, Germany) and filtered through a
0.22 µm mesh filter (MILLIPORE, Burlington, United States).
After the final dilutions were ready, the bottles were closed
and subjected to automatic succussion in a mechanical arm
(Denise—AUTIC, São Paulo, Brazil), following the national
standards.21
Three types of controls were used. The first was made
with purified sterile water submitted to succussion,
the second consisted solely of purified sterile water, with-
out succussion, and the third was prepared from the
centesimal homeopathic potency of the vehicle in sterile
water, here named Ethilicum 1cH, containing 0.7% of alco-
hol. The use of three different controls permitted the
identification of any effects associated with the vehicles
under serial agitation.
All preparation and storage procedures and the handling
of all medication were performed under sterile conditions in
a laminar flow hood.
Bottle Coding
The bottles of MC working potencies and Ethilicum 1cH had
their labels exchanged by a technician not involved in the
experiment before the study began. The original labels and
the respective codes were kept in a sealed envelope
throughout the test procedures and was opened only after
the end of the statistical analysis. Thus, all tests were
performed blind.
 Mercurius Corrosivus and Bioresilience of Artemia Salina
Hatching of Artemia Salina Cysts
The experimental manipulation for hatching of nauplii fol-
lowed these steps:
1. Preparation of artificial sea water
One liter of artificial sea water was prepared by adding 30 g
of artificial sea salt (Ocean Fish—PRODAC, Citadella PD, Italy) in
1.0 L of distilled water, under constant agitation. Then, food
was included in a proportion of 5.0%. The food consisted of a
suspension of powdered biological yeast (Saccharomyces cer-
evisiae) (5 mg/mL), previously prepared in artificial seawater.
2. Definition of the experimental groups
Nine 96-well plates were used weekly, within four weeks
of experiment. Thus, 36 plates were used throughout the
study. For each plate, treatments were dispensed in rows of
12 wells, each containing cysts in the process of hatching,
according to the following design:
• Cysts not challenged with HgCl2 and untreated.
• Cysts challenged with HgCl2 and treated with pure sterile
water.
• Cysts challenged with HgCl2 and treated with succussed
pure sterile water.
• Cysts challenged with HgCl2 and treated with Ethilicum
1cH.
• Cysts challenged with HgCl2 and treated with MC 6cH.
• Cysts challenged with HgCl2 and treated with MC 30cH.
• Cysts challenged with HgCl2 and treated with MC 200cH.
The cysts (Global Biotérios, São Paulo, Brazil) were kept
dry (290,000 units per gram) in an airtight desiccator with
reduced humidity, containing a layer of silica gel at the
bottom. Immediately before use, the cysts were homo-
geneously suspended in artificial seawater (75 mg of cysts
per 200 mL) to obtain six to eigth cysts per 100 µL. This was
the volume inserted in each well.
3. Challenging of cysts with mercury chloride
Mercury chloride was inserted into the water at the same
time as the cysts at a concentration equivalent to LC 10
(lethal concentration 10%) or 5.0 µg/mL (containing 370 µM
of total mercury—THg—per well), as previously established
in a pilot experiment. A low (but harmful) concentration of
mercury (LC 10) was chosen for this experiment to mimic
usual conditions of mild environmental contamination as-
sociated with processes of chronic toxicity.7,62 Aliquots of MC
6, 30 or 200cH were inserted in each well at the time of
intoxication, at a volume equal to 10% of the final volume of
sea water. Thus, the final concentration of residual alcohol in
each well was 0.07%, not enough to promote biological effects
on the brine shrimps.63
4. Analysis of hatching rate and nauplii viability
After 24 and 48 hours, each well was observed using a digital
pen microscope (Digital Microscope with Zoom 1000x R Camera
2.0 Mega Pixels USB 6 Leds, Beijing, China), and data were
recorded (AMCAP, Beijing, China) on a computer (Yoga520,
Lenovo, Brazil) for counting of hatched/non-hatched cysts.
5. Experimental design and environment control
Plating was performed on three consecutive days (three
series in triplicates) in a week, for 4 weeks, totaling 36
repetitions and 14,468 hatched nauplii for analyses.
Homeopathy
Pinto et al. 247
The plates containing the cysts were placed in a hand-
made Faraday cage and illuminated 24 hours a day with 50-
60 Hz LED light (IT-666, Yi Teng, China) to promote hatching
in a stable environment. They remained open to allow
enough oxygenation of water surface, necessary to promote
hatching. The laboratory environmental conditions were
monitored throughout the experimental period. The room
temperature average was 22.83  0.43°C and the electromag-
netic flow near the plates was 0.07  0.06 µT at 50Hz (Smart-
Sensor Intel Instruments, AS 1392, Singapore).
Moon phase was also observed during the whole experi-
mental period and any statistical correlations between moon
phases and hatching rates were noted. All moon phases were
included within this study period.
After recordings, made at 24 and 48 hours of incubation,
the plates were frozen, and the contents stored for later
physicochemical analysis.
Physicochemical Analysis of Water
Scanning Electron Microscopy and X-Ray Dispersive
Energy Spectroscopy (JEOL 6510)
The contents of wells were collected from randomly select-
ed plates and grouped into sample pools for each type of
treatment (controls or MC 6cH, MC 30cH, MC 200cH), in a
manner such that each treatment was evaluated in tripli-
cate. Each pool was centrifuged at 10,000 RPM (11,068 g,
Centrifuge MCA mod.1–4K, Sigma, Osterode am Harz,
Germany) for 60 minutes, to separate the biological sedi-
ment from water. Then, the supernatant was filtered
through a 0.22 µm mesh filter (Millipore, Burlington, United
States) and centrifuged again at 10,000 RPM (11,068 g) for
1 hour, to obtain non-biological particulate sediment and
the respective supernatant, which was stored for measure-
ment of soluble mercury. At the end of this process, two
kinds of sediment (biological and inorganic) were used in
scanning electron microscopy/X-ray dispersive energy spec-
troscopy (SEM/EDS) analysis.
The analysis of inorganic sediment was performed using
round basement coverslips cleaned with acetone and dried.
They were previously metallized in a vacuum chamber
(Desk V, Denton Vacuum, Moorestown, New Jersey, United
States) with a gold foil, and 10 µL of each sediment removed
from the bottom, just after centrifugation, was placed on the
coverslip surface as droplets, to avoid the liquid spreading.
The coverslips were then placed in an oven at 50°C until the
droplets dried. The analyses were made in a scanning elec-
tron microscope (JSM 6510, JEOL, Tokyo, Japan) with the EDS
system by a primary beam of accelerated electrons set at
15kV.
For the analysis of the biological sediment, the samples
were fixed in 3% buffered formaldehyde for 24 hours at 4°C,
washed in distilled water, and dehydrated in an increasing
alcoholic graduation bath, starting from 30%, for 5 minutes at
each graduation until finally reaching three baths in ethanol
100%. Then they were taken to the critical point in HMDS
(hexamethyldisilazane, Sigma-Merck, United States) with
three immersions of 5 minutes. After total drying, the
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248 Mercurius Corrosivus and Bioresilience of Artemia Salina Pinto et al.

samples were placed in round glass coverslips (22 mm)
adhered to each stub. The metallization of biological samples
was performed in a vacuum chamber (as described above),
using a gold foil. The images were captured by SEM at 1000x
magnification and five random fields per sample were
recorded. EDS was used to identify the chemical elements
present. Silica and gold were not considered for evaluation to
avoid bias, as these same elements were part of the metal-
lized coverslip structure.
Similarly, samples of biological sediment deposited on
previously metallized coverslips were analyzed by area. The
droplet area delimited for analysis comprised a 2.5 mm  3.5
mm square, covering at least 80% of the material surface. In
this case, samples were analyzed at 30x magnification, using
the same equipment and conditions described
above. ►Fig. 1A and 1B show typical SEM images of non-
biological and biological sediments, respectively.
Determination of Total Mercury (THg) by Thermal
Decomposition, Amalgamation and Atomic Absorption
using a Direct Hg Analyzer
The measurement of soluble mercury was performed in a DMA-
80 Hg analyzer (Milestone, Sorisole, Italy). The mercury content
was determined based on thermal decomposition, amalgam-
ation and atomic absorption.41 A small amount of filtered water
sample (20–200 μL) was placed in each quartz boat, which was
then introduced into an oven enriched with oxygen, and heated
to 750°C, to proceed the thermal decomposition.
In this method, halogens, nitrogen oxides and sulfur
released in decomposition are retained in a catalyst. Mercury
is selectively amalgamated in an amalgamation cell, where
mercury is separated from any decomposition products.
Finally, the amalgamator is heated quickly, releasing the
mercury vapor to be quantified by atomic absorption.
The LD detection limit obtained was 0.4 μg/L; the preci-
sion and accuracy of the measurement was confirmed by the
recovery of 90% of the total mercury concentration with a
certified reference material, DORM-4, as described by Pecor-
aro et al.42 The high accuracy of this method allows repro-
ducibility and reliability of analysis even in small samples.
One measurement was initially made for each sample.
Those samples presenting discrepant values in relation to the
median (12.45 ppm) were selected to be measured in differ-
ent replications, to check their validity.
Interaction of MC Potencies with Solvatochromic Dyes
Samples of MC working potencies were submitted to spec-
trophotometry using different solvatochromic dyes. Dyes
were chosen to cover all the visible spectra, in order to
find the most sensitive of MC potencies (►Supplementary
Table S2, available online only). The dyes were diluted in
absolute alcohol on the evening prior to use and stored in
sterile amber glass flasks at room temperature, and isolated
from any strong electromagnetic fields.
Manipulation of dyes and cuvettes was performed in a
laminar flux hood. Samples and dye solutions were filtered in
a 0.22 µm mesh filter (Millipore, Burlington, United States) just
before use. The ratio between dye solution and sample in the
cuvettes was 60:1. The internal controls consisted of succussed
or non-succussed sterile purified water. Readings were per-
formed using a UV-vis spectrophotometer (FEMTO 800 XI, São
Paulo, Brazil). The absorbance of each dye solution was scanned
between 350 and 800 nm, with a resolution of 1 nm, to find the
exact wavelength corresponding to the peak of absorbance.
Analyses were performed at the wavelength corresponding to
this peak. All analyses were done in triplicate. Purified water
was used as the absorbance baseline, to calibrate the equipment
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
Fig. 7 Absorbance of the dye ET33 using visible light spectropho-
tometry after adding different MC dilutions and controls.
(F (5,10) ¼ 9.137, p ¼ 0.0017, partial ɳ 2 ¼ 0.820); p ¼ 0.04 in relation to
the other groups, according to Tukey’s post-test.
Interaction of Homeopathic Samples with
Solvatochromic Dyes
The interaction of homeopathic potencies with solvatochro-
mic dyes was selective: only MC 30cH showed interaction
with ET 33, by reducing absorbance in relation to the controls
(►Fig. 7). The other dyes did not show any interaction with
MC dilutions.
Discussion
The results obtained in this study show the effects of
various preparations of MC in modulating the toxicity of
HgCl2 LC 10 (lethal concentration of 10%) exposure in
relation to hatching of Artemia salina nauplii. The effect
of MC 30cH was the most significant and appeared to be
independent of external variables such as the moon phases
and the time of observation.
Exposure to HgCl2 reduced the hatch rate, indicating a
defense process of the cysts. This non-specific effect can be
hypothetically attributed to oxidative stress on the em-
bryo,64 due to the presence of nano- and air microbubbles
(oxygen) and/or the presence of hydroxyl radicals generated
during the succussion process of a polar medium.17,18,38
Cysts are forms of resistance of the species and their perma-
nence as such reveals a well-known resilience mechanism,
involving the production of chaperone (p26, ArHsp21,
ArHsp22, and artemin) and other proteins by the em-
bryo.65–67 Similarly, the treatment with Ethilicum 1cH (suc-
cussed 0.7% alcoholic solution) was also related to a first level
of cyst protection. According to Sandri et al, potentized
alcohol cannot be considered as an inert vehicle, but a
specific homeopathic medicine, thus a comparative
substance.68
This is part of an adaptation process used by Artemia spp.,
called “diapause”, to preserve the species when exposed to
Homeopathy
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-
tent population effect.
The physicochemical analyses revealed an increase of
soluble THg in samples treated with MC 30cH, and interac-
tivity of MC 30cH itself to a specific solvatochromic dye (ET
33) was also observed (►Fig. 7). Solvatochromic dyes are
very sensitive to the presence of homeopathic preparations,
producing subtle but precise changes in the peak of absor-
bance,43–47 and different patterns of reactivity may corre-
spond to different biological activities47 through changes in
the polarity of the medium (solutes and/or water). According
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-
tact of dyes with water structures and aggregates, suggesting
that these dyes, in the presence of homeopathic potencies,
are more electrochromic than solvatochromic.46 Thus,
effects related to mere biochemical interactions between
additional ions present in water bulk and these dyes are
unlikely.
Hypothetically, the MC 30cH effects on hatching, THg
soluble concentration and ET33 dye spectra could be associ-
ated with a common biophysical mechanism, possibly relat-
ed to a particular excitation state of the liquid medium.
The results allow for the use of these homeopathic
potencies as agents in a simple and low-cost remediation
procedure, able to improve natural bioresilience processes
and be monitored by solvatochromic dyes, even in a large
volume of water, as previously reported.48
1Circalunar variations are known to be important modula-
tion factors on the physiological processes of several marine
species. In 2020, Andreatta and Tessmar-Raible pointed out
connections between rhythms of metabolic and endocrine
pathways and circalunar variations,57 making it possible to
identify molecular markers, such as gonadotrophin-releasing
hormone and neuropeptides, related to the expression of
“clock genes” in several marine species.73 Lunar cycles also
promote oscillation and synchronization of other biological
processes, such as general activity, photosensitivity, and verti-
cal migration of zooplankton.55,57,73 Environmental variables,
Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
252 Mercurius Corrosivus and Bioresilience of Artemia Salina
such as the moon phase, also seem to be involved in Artemia
salina biological processes, as shown herein, but they were not
able to change the specific effects of MC 30cH.
Artemia salina seemed to be a good study model for the
formulation of strategies to mitigate the impact of water
contamination on local living systems,70 by improving their
resilience to harmful stimuli. MC 30cH seems to optimize
this natural resilience, suggesting it as a potential adjuvant
tool to reduce the environmental impact of residual levels of
mercury, especially if applied in association with traditional
processes of clearance of heavy metals from water.10–15 This
conclusion is in accordance with previous studies on homeo-
pathic potencies that often show a constant pattern of action:
the facilitation of adaptive processes of the organism against
aggressive agents in a non-linear way,74–80 involving the
modulation of immunological and metabolic processes, as
well as gene expression.27–35 The findings described here
follow these same pro-adaptative features.
Another aspect that deserves attention is the property of
nanobubbles to present electrically charged interfaces, adsorb-
ing suspended solids in seawater, which could suggest that the
simple insertion of water subjected to succussion into seawater
could trigger some non-specific purification process.38,81–83
Moreover, the oxygen contained in nanobubbles reduces the
synthesis of methylmercury in water.19,20
An alternative explanatory hypothesis for the effects of
MC 30cH would be the stock of oxygen and chlorine in such
deposits, able to be released into water by diffusion. Chlorine
could be a source of mercury stabilization in water, in the
form of HgCl2 or CH3HgCl,82 preventing mercury’s absorp-
tion by the exposed nauplii and reducing its toxicity. This
hypothesis is reinforced by the fact that MC 30cH was the
only treatment related to higher concentration of soluble
THg in water, compared with the other treatments.
In all cases, after 48 hours, when almost 100% of cysts were
hatched, the level of THg in water was 200 times lower than the
content inserted into the wells. A certain degree of absorption
of HgCl2 by yeasts (used as food for nauplii) associated with the
natural volatilization of HgCl214,83,84 could be the reason of
this decay, since there was no detectable deposition of mercu-
ry in organic and inorganic microaggregates.
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
by the nauplii. These animals can perform “osmoregulation”
using the “neck organ”, a Cl-excretory gland, which gives to
Artemia salina the ability to adapt very well to hyper-saline and
harsh environments. This gland works by means of the enzyme
Naþ/Kþ-ATPase, whose activity depends on aerobic metabo-
lism.52 The suggested hypothesis to explain the effects of the
MC 200cH, in this case, would be an increase in the activity of
the “neck organ”, by mechanisms still unknown.
The protective activity of MC against intoxications is
described in previous studies. Jäger et al, in 2019, observed
protective effects of specific homeopathic potencies of mer-
cury chloride in Lemna gibba L. intoxicated by the same
substance.85 Likewise, the protective action of specific
Homeopathy Vol. 110 No. 4/2021 © 2021. The Faculty of Homeopathy. All rights reserved.
Pinto et al.
homeopathic potencies of mercury against heavy metal
poisoning is also reported in rodents and macrophages
in vitro.30,31 These results suggest that the use of isother-
apeutic preparations to mitigate the effects of water pollu-
tion on living systems could be a plausible tool in an eventual
environmental management program and be proposed as a
new approach for sustainable and low-cost health promotion
for aquatic organisms32,33 that are exposed to low, but
constant, concentrations of mercury.81,86 Other studies per-
formed by our group also point to similar effects, using the
same methods to study other toxins.87,88
Conclusion
The exposure of Artemia salina cysts to HgCl2 inserted in
seawater in a lethal concentration of 10% (LC10) leads to
delay of hatching—a natural adaptative process of the species
to survive in harmful environments. The addition of MC 30cH
into seawater led to a significant enhancement of this
response, independently of external variables.
The THg distribution among living and non-living com-
partments in the aquatic medium and the interactivity of MC
30cH with ET33 dye can each help to build hypotheses to
explain this peculiar biological effect.
The study presents the potential use of isotherapy as a
sustainable tool for bioresilience management in cases of
water contamination.
Highlights
• Mercurius corrosivus (MC) was investigated as a puta-
tive bioremediatory agent.
• Cysts of Artemia salina exposed to low concentrations of
HgCl2 were used as experimental model.
• The dilution MC 30cH potentiated the delay of cysts
hatching, an adaptative process.
• It changed the balance of soluble THg, chlorine and
oxygen contents in water.
• It seems to change medium polarity, as indicated by its
interaction with solvatochromic dyes.
• MC 30cH is proposed as a potential tool for improving
bioresilience to mercury.
Supplementary Files
Supplementary Table S1 Pilot experiment to determine
the correspondence between HgCl2 concentrations and
48-hour-old nauplii lethality
Supplementary Table S2 Standardized dyes used in this
experiment
Supplementary Fig. S1 Profile of the spectrum of EDS
analyses, from mineral sediment obtained by centrifu-
gation of filtered sea water.
Conflict of Interest
None declared.
 Mercurius Corrosivus and Bioresilience of Artemia Salina
Acknowledgements
We are grateful to the Pharmacy HN Cristiano, São Paulo,
and Dr. Amarylis Toledo César, the responsible Pharmacist,
for manufacturing the study’s Mercurius corrosivus, which
was prepared from the salt purchased from Sigma-Merck.
We thank the University Paulista (UNIP) for the scholar-
ships of Andreia A.G. Pinto, Michelle S. Aguiar and Sandra A.
G. Pinto, and CAPES-Prosup for the scholarship of Ednar do
Nascimento Coimbra, Mirian Y.D. Nagai and Suham Now-
rooz. Steven Cartwright gratefully acknowledges funding
from Standard Homeopathic Company/Hylands, United
States. We also thank Wilton Pereira dos Santos for techni-
cal support in the aquatic organism laboratory.
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