ABSTRACT
Cadmium
is recognize has a toxicant to both human and it’s environment and recent
investigations have shown its level of toxicity in association in liver
damage. The aim of study is to determine
the effect of calcium tainted water on cadmium induced liver damage have been examine in this studies, 20 female
wistar rat were used in this study the rats divided into four groups each
containing 5 rats per group . The group one was maintained on normal feed and
water only, the group 2 were exposed to cadmium only while the group three were
exposed to calcium only and the group four were exposed simultaneously to
cadmium and calcium. Each of the animal was given treatment based on their body
weight (0.9 mg per kg body weight). The treatments were administered to the
animals once a day for two weeks. At the end of two weeks the animals were
sacrificed and the following biochemical markers were measured; Alkaline
phosphatase, total protein, alanine amino transferase, total bilirubin and
direct bilirubin. All the biochemical
markers were negatively affected by cadmium with exception to Albumin and total
protein. The study reveal that cadmium has the potential to induce
hepatotoxicity and calcium tainted water offer little ameliorating affect to
cadmium induce liver damage.
TABLE OF CONTENTS
Cover
page
Certification……………………………………………………………i
Dedication……………………………………………………………..ii
Acknowledgement…………………………………………………….iii
Table
of contents………………………………………………………iv
Abstracts………………………………………………………………vii
CHAPTER ONE
Introduction
and literature review…………………………………….1
Cadmium………………………………………………………………3
Physical
and chemical properties……………………………………..4
Occurance………………………………………………………………5
Biological
role………………………………………………………….6
Cadmium
poisoning……………………………………………………7
Calcium………………………………………………………………..11
Calcium
compound……………………………………………………12
Nutrition
………………………………………………………………14
Bone
health…………………………………………………………….16
Cardiovascular
impact…………………………………………………16
Hazard
and toxicity……………………………………………………….17
Hard
water …………………………………………………………………18
Sources
of hardness ……………………………………………………….18
Temporary
hardness………………………………………………………..19
Permanent
hardness………………………………………………………..20
Effect
of hard water…………………………………………………………21
Health
consideration………………………………………………………22
The
liver…………………………………………………………………...23
Anatomy
of the liver……………………………………………………..24
Diseases
of the liver………………………………………………………29
Liver
function test………………………………………………………30
CHAPTER TWO
Materials
and methods…………………………………………………35
Materials……………………………………………………………….35
Animals……………………………………………………………….36
Preparation
of contaminated water………………………………….36
Treatment
of animal…………………………………………………37
Preparation
of seral sample…………………………………………..37
Preparation
of tissue homogenates………………………………….37
Biochemical
analyses………………………………………………….38
CHAPTER THREE
Results………………………………………………………………….46
CHAPTER FOUR
Discussion……………………………………………………………….53
Conclussion……………………………………………………………..54
References
……………………………………………………………...55
Appendix
i………………………………………………………………66
Appendix
ii……………………………………………………………..68
Appendix
iii…………………………………………………………… 70
Appendix
iv…………………………………………………………….75
Appendix
v……………………………………………………………..79
CHAPTER ONE
1.0
INTRODUCTION AND LITERATURE REVIEW
Heavy
metals are toxic agent. They are toxic to humans and animals. Heavy metals
which establishes toxic actions to humans include; cadmium (Stohs and Bagchi,1995),
lead ( Ferner, 2001) and mercury (Hawkes, 1997). Each of these has been
studied in isolation for toxicity
(Huton and Symon, 1986; Nriagu and Pacyna, 1988; Nriagu, 1989). But, in the
eco-system, be it air, atmosphere, land, and water where they occur, they do
not exist in isolation. They occur in close association with other metal and
non-metallic elemental pollutants. Among the metallic pollutant could be
calcium, copper, zinc, magnesium, manganese, iron and others. Metals are known to interact with one
another. The interaction can bring two elements together in close proximity or
it could cause out right displacement of one another. When ingested together in
food and water, they antagonize each other. When it comes to intestinal and
pulmonary absorption, it is therefore conceivable that the presence of other
elements can the toxic potential of each of the heavy metals that have been
studied in isolation.
Eborge (1994) reported that warri
river has an unacceptable high cadmium level, 0.3 mg cadmium per liter of water
which was 60 folds above the maximum allowable level of 0.005 mg per liter.
This report prompted our earlier studies on the hepato, nephro and gonadial
toxicity of cadmium. In rats exposed to this high dose via water and diet, the
diet was formulated with feed exposed to 0.3 mg cadmium per water. In the ambient
water as protein source and the toxic effect investigated and reported (Asagba
and obi 2000; Asagba and Obi 2001; Obi and Ilori 2002; Asagba and Obi 2004a;
Asagba and Obi 2004b; Asagba and Obi 2005).The study focus on cadmium without
taking into consideration the fact that other metals were also present in the
river water, and as such were co-consumed by the communities using the river
water for cooking drinking and for other domestic purposes. Hence, it is
desirable to know if the presence of other metals would enhance or diminish the
toxic potential of cadmium or indeed if any other heavy metals such as lead
that was mentioned above. Therefore, the aim of the present study was to
re-examine the toxic potential of cadmium in the presence of other metals such
as calcium and magnesium.
The objectives set out to achieve
were;
1. Re-examination
of toxicity of using established and those for liver toxicity namely; blood
alanine amino transferase and aspartate amino transferase, alkaline
phosphatase, bilirubin, albumin and total protein.
2. Re-examine
the status parameter in the absence of cadmium but in the presence of calcium
or magnesium or both.
3. Re-examine
this parameters in the presence of cadmium, calcium and magnesium.
1.1 CADMIUM
Cadmium is a chemical
element with symbol Cd
and atomic number 48. This soft,
bluish-white metal is chemically similar to the two other stable metals in group 12, zinc and mercury. Like zinc, it prefers oxidation state +2 in most of its
compounds and like mercury it shows a low melting point compared to transition metals. Cadmium and its congeners are not always
considered transition metals, in that they do not have partly filled d or f
electron shells in the elemental or common oxidation states. The average
concentration of cadmium in Earth's crust is between 0.1 and 0.5 parts per
million (ppm). It was discovered in 1817 simultaneously by Stromeyer and Hermann, both in
Germany, as an impurity in zinc
carbonate. Cadmium occurs as a minor component in most zinc
ores and therefore is a byproduct of zinc production. It was used for a long
time as a pigment and for corrosion-resistant plating on steel,
whereas cadmium compounds were used to stabilize plastic.
The use of cadmium is generally decreasing due to its toxicity
(it is specifically listed in the European Restriction of Hazardous
Substances (Morrow, 2010)) and the replacement of nickel-cadmium batteries with nickel-metal hydride and
lithium-ion batteries. One of its
few new uses is in cadmium
telluride solar
panels. Although cadmium has no known biological function in
higher organisms, a cadmium-dependent carbonic anhydrase has been found
in marine diatoms.
1.1.1 PHYSICAL PROPERTIES
Cadmium
is a soft, malleable,
ductile,
bluish-white divalent
metal.
It is similar in many respects to zinc but forms complex compounds (Holleman et al., 1985). Unlike other metals,
cadmium is resistant to corrosion and as a result it is used as a protective layer
when deposited on other metals. As a bulk metal, cadmium is insoluble
in water and is not flammable; however, in its powdered form it may
burn and release toxic fumes (CSEM, 2011).
1.1.2 CHEMICAL PROPERTIES
Although
cadmium usually has an oxidation state of +2, it also exists in the +1
state. Cadmium and its congeners are not always considered
transition metals, in that they do not have partly filled d or f electron
shells in the elemental or common oxidation states (Cotton, 1999). Cadmium
burns in air to form brown amorphous cadmium oxide
(CdO); the crystalline form of this compound is a dark red
which changes color when heated, similar to zinc oxide.
Hydrochloric acid, sulfuric acid
and nitric acid
dissolve cadmium by forming cadmium
chloride (CdCl2), cadmium
sulfate (CdSO4), or cadmium
nitrate (Cd(NO3)2). The oxidation state +1 can
be reached by dissolving cadmium in a mixture of cadmium chloride and aluminium chloride, forming the Cd22+
cation, which is similar to the Hg22+ cation in mercury(I) chloride (Holleman et al., 1985).
Cd + CdCl2 + 2 AlCl3
→ Cd2(AlCl4)2
The
structures of many cadmium complexes with nucleobases,
amino acids
and vitamins
have been determined (Carballo et al.,
2013).
1.1.3 OCCURRENCE
Cadmium
metal
Cadmium makes up about 0.1 ppm
of Earth's crust. Compared with the more
abundant 65 ppm zinc, cadmium is rare (Wedepohl, 1995). No significant
deposits of cadmium-containing ores are known. Greenockite
(CdS), the only cadmium mineral
of importance, is nearly always associated with sphalerite
(ZnS). This association is caused by the geochemical similarity between zinc
and cadmium which makes geological separation unlikely. As a consequence,
cadmium is produced mainly as a byproduct from mining, smelting, and refining
sulfidic ores of zinc, and to a lesser degree, lead and copper. Small amounts of cadmium, about 10% of
consumption, are produced from secondary sources, mainly from dust generated by
recycling iron and steel scrap. Production in the United States began in 1907,
(Ayres et al., 2003) but it was not
until after World War I that cadmium came into wide use (Plachy, 1998). One
place where metallic cadmium can be found is the Vilyuy
River basin in Siberia
(Fthenakis, 2004).
Rocks mined to produce phosphate fertilizers contain varying
amounts of cadmium, leading to a cadmium concentration of up to 300 mg/kg
in the produced phosphate fertilizers and thus in the high cadmium content in
agricultural soils (Grant and Shepperd , 2008). Coal can contain significant
amounts of cadmium, which ends up mostly in the flue dust (Bettinelli et al., 1988).
1.1.4 BIOLOGICAL ROLE
Cadmium has no known useful role in higher organisms, (Hogan,
2010) but a cadmium-dependent carbonic anhydrase has been found
in some marine diatoms
(Lane et al., 2005). The diatoms live in environments with very low zinc
concentrations and cadmium performs the function normally carried out by zinc
in other anhydrases. The discovery was made using X-ray absorption fluorescence
spectroscopy (XAFS) (Lane et al.,
2000).
The highest concentration of cadmium has been found to be
absorbed in the kidneys of humans, and up to about 30 mg of cadmium is
commonly inhaled throughout childhood and adolescence (Perry et al., 1976). Cadmium can be used to
block calcium channels in chicken neurons (Swandulla and Armstrong, 1989).
Analytical methods for the determination of cadmium in biological samples have
been reviewed (klorz et al., 2013).
1.1.5 ENVIRONMENT
The biogeochemistry of cadmium and its release to the
environment has been the subject of review, as has the speciation of cadmium in
the environment (Cullen et al., 2013).
1.1.6 CADMIUM
POISONING
The
bioinorganic aspects of cadmium toxicity have been reviewed (Maret et al., 2013).The most dangerous form of
occupational exposure to cadmium is inhalation of fine dust and fumes, or
ingestion of highly soluble cadmium compounds. Inhalation of cadmium-containing fumes can
result initially in metal fume fever but may progress to chemical pneumonitis,
pulmonary
edema, and death (Hayes, 2007). Cadmium is also an environmental
hazard. Human exposures to environmental cadmium are primarily the result of
fossil fuel combustion, phosphate fertilizers, natural sources, iron and steel
production, cement production and related activities, nonferrous metals
production, and municipal solid waste incineration. Bread, root crops, and vegetables also
contribute to the cadmium in modern populations (Mann, 2012). There have been a
few instances of general population toxicity as the result of long-term
exposure to cadmium in contaminated food and water, and research is ongoing
regarding the estrogen mimicry that may induce breast cancer (Mann, 2012). In
the decades leading up to World War II, mining operations contaminated
the Jinzū River
in Japan with cadmium and traces of other toxic metals. As a consequence,
cadmium accumulated in the rice crops growing along the riverbanks downstream
of the mines. Some members of the local agricultural communities consuming the
contaminated rice developed itai-itai disease and renal abnormalities, including proteinuria
and glucosuria
(Nogawa et al., 2004).
Jinzū River
area, which was contaminated with cadmium
The
victims of this poisoning were almost exclusively post-menopausal women with
low iron and other mineral body stores. Similar general population cadmium
exposures in other parts of the world have not resulted in the same health
problems because the populations maintained sufficient iron and other mineral
levels. Thus, although cadmium is a major factor in the itai-itai disease in
Japan, most researchers have concluded that it was one of several factors.
Cadmium is one of six substances banned by the European Union's Restriction on Hazardous Substances
(RoHS) directive, which bans certain hazardous substances in electrical and
electronic equipment but allows for certain exemptions and exclusions from the
scope of the law. The International Agency for Research on Cancer has
classified cadmium and cadmium compounds as carcinogenic to humans. Although
occupational exposure to cadmium is linked to lung and prostate cancer, there
is still a substantial controversy about the carcinogenicity of cadmium in low,
environmental exposure. Recent data from epidemiological studies suggest that
intake of cadmium through diet associates to higher risk of endometrial, breast
and prostate cancer as well as to osteoporosis in humans (Julin et al., 2012). A recent study has
demonstrated that endometrial tissue is characterized by higher levels of
cadmium in current and former smoking females (Rzymski et al., 2014). Although some epidemiological studies show a
significant correlation between cadmium exposure and occurrence of disease
conditions in human populations, a causative role for cadmium as the factor
behind these effects remains yet to be shown. In order to prove a causative
role, it will be important to define the molecular mechanisms through which
cadmium in low exposure can cause adverse health effects. One hypothesis is
that cadmium works as an endocrine disruptor because some experimental
studies have shown that it can interact with different hormonal
signaling pathways. For example, cadmium can bind to the estrogen
receptor alpha, (Fechner et
al., 2011) and affect signal transduction along the estrogen
and MAPK
signaling pathways at low doses (Ali et
al., 2010).
Tobacco
smoking is the most important single source of cadmium exposure in the general
population. It has been estimated that about 10% of the cadmium content of a
cigarette is inhaled through smoking. The absorption of cadmium from the lungs
is much more effective than that from the gut, and as much as 50% of the
cadmium inhaled via cigarette smoke may be absorbed (Friberg, 1983). On
average, smokers have 4–5 times higher blood cadmium concentrations and 2–3
times higher kidney cadmium concentrations than non-smokers. Despite the high
cadmium content in cigarette smoke, there seems to be little exposure to
cadmium from passive smoking. No significant effect on blood
cadmium concentrations has been detected in children exposed to environmental
tobacco smoke. In the non-smoking part
of the population food is the biggest source of exposure to cadmium. High
quantities of cadmium can be found for example in crustaceans, molluscs,
offals, and algal products. However, due to the higher consumption the most
significant contributors to the dietary cadmium exposure are grains,
vegetables, and starchy roots and tubers. Cadmium exposure is a risk factor
associated with early atherosclerosis and hypertension, which can both lead to
cardiovascular disease (Jarup, 1998).
1.2 CALCIUM
Calcium is a chemical
element with symbol Ca and atomic number 20.
Calcium is a soft gray alkaline earth metal, and is the
fifth-most-abundant element by mass
in the Earth's crust.
Calcium is also the fifth-most-abundant dissolved ion in seawater by both molarity
and mass, after sodium,
chloride, magnesium,
and sulfate (Dickson and Goyet 1994). Calcium is
essential for living organisms,
in particular in cell
physiology, where movement of the calcium
ion Ca2+ into and out of the cytoplasm
functions as a signal for many cellular processes. As a major material used in
mineralization of bone, teeth
and shells, calcium is the most abundant metal
by mass in many animals.
1.2.1 CALCIUM
COMPOUNDS
- Calcium carbonate
(CaCO3) is used in manufacturing cement
and mortar, lime, limestone (usually used in the steel
industry) and aids in production in the glass industry. It also has
chemical and optical uses as mineral specimens in toothpastes, for example.
- Calcium hydroxide
solution (Ca (OH)2) (also known as limewater)
is used to detect the presence of carbon dioxide by being bubbled through
a solution. It turns cloudy where CO2 is present.
- Calcium arsenate
(Ca3 (AsO4)2) is used in insecticides.
- Calcium
carbide (CaC2) is used to make acetylene gas (for use in acetylene torches for welding)
and in the manufacturing of plastics.
- Calcium chloride
(CaCl2) is used in ice
removal and dust
control on dirt roads, in conditioner for concrete,
as an additive in canned tomatoes,
and to provide body for automobile
tires.
- Calcium
citrate (Ca3 (C6H5O7)2)
is used as a food preservative.
- Calcium
cyclamate (Ca (C6H11NHSO3)2)
is used as a sweetening agent in several countries. In the United States
it is no longer permitted for use because of suspected cancer-causing
properties (Newton, 2009).
- Calcium gluconate
(Ca (C6H11O7)2) is used as a food additive and in vitamin pills.
- Calcium hypochlorite
(Ca(OCl)2) is used as a swimming
pool disinfectant,
as a bleaching agent, as an ingredient in deodorant, and in algaecide and fungicide.
- Calcium permanganate
(Ca (MnO4)2) is used in liquid rocket propellant, textile production, as a water sterilizing
agent and in dental procedures.
- Calcium phosphate
(Ca3(PO4)2) is used as a supplement for animal feed, fertilizer,
in commercial production for dough
and yeast products, in the manufacture of glass, and in dental products.
- Calcium phosphide
(Ca3P2) is used in fireworks,
rodenticide,
torpedoes and flares.
- Calcium stearate
(Ca(C18H35O2)2) is used in the
manufacture of wax crayons, cements,
certain kinds of plastics
and cosmetics, as a food additive, in the production
of water resistant materials and in the production of paints.
- Calcium
sulfate (CaSO4·2H2O)
is used as common blackboard chalk, as well as, in its hemihydrate form
better known as Plaster of Paris.
- Calcium tungstate
(CaWO4) is used in luminous paints,
fluorescent lights and in X-ray studies.
- Hydroxylapatite
(Ca5 (PO4)3(OH), but is usually written
Ca10 (PO4)6(OH)2) makes up
seventy percent of bone.
Also carbonated-calcium deficient hydroxylapatite is the main mineral of
which dental
enamel and dentin
are comprised.
1.2.2 NUTRITION
Calcium is an important component of
a healthy diet and a mineral necessary for life. The National
Osteoporosis Foundation says, "Calcium plays an
important role in building stronger, denser bones early in life and keeping
bones strong and healthy later in life." Approximately 99 percent of the
body's calcium is stored in the bones and teeth. The rest of the calcium in the
body has other important uses, such as some exocytosis,
especially neurotransmitter release, and muscle contraction. In the electrical conduction system
of the heart, calcium replaces sodium as the mineral that
depolarizes the cell, proliferating the action potential. In cardiac muscle,
sodium influx commences an action potential, but during potassium efflux, the
cardiac myocyte experiences calcium influx, prolonging the action potential and
creating a plateau phase of dynamic equilibrium. Long-term calcium deficiency can lead to rickets and poor blood
clotting and in case of a menopausal woman, it can lead to osteoporosis, in which the bone
deteriorates and there is an increased risk of fractures. While a lifelong
deficit can affect bone and tooth formation, over-retention can cause hypercalcemia (elevated levels of calcium
in the blood), impaired kidney function and decreased absorption of other
minerals (Catharine et al., 2011).
Several sources
suggest a correlation between high calcium intake (2000 mg per day, or
twice the U.S. recommended daily allowance,
equivalent to six or more glasses of milk per day) and prostate cancer (giovannucci et al., 1998). Dairy
products, such as milk and cheese, are a well-known source of
calcium. Some individuals are allergic to dairy products and even more people,
in particular those of non Indo-European descent, are lactose-intolerant, leaving them
unable to consume non-fermented dairy products in quantities larger than about
half a liter per serving. Others, such as vegans,
avoid dairy products for ethical and health reasons. Many good vegetable
sources of calcium exist, including seaweeds
such as kelp, wakame
and hijiki; nuts and seeds like almonds, hazelnuts,
sesame, and pistachio;
blackstrap molasses; beans
(especially soy beans); figs; quinoa; okra; rutabaga; broccoli;
dandelion leaves; and kale.
In addition, several foods and drinks, such as orange
juice, soy milk,
tofu,
breakfast cereals, and breads are often fortified with calcium. Numerous
vegetables, notably spinach,
chard
and rhubarb have a high calcium content, but they
may also contain varying amounts of oxalic acid
that binds calcium and reduces its absorption. The same problem may to a degree
affect the absorption of calcium from amaranth,
collard greens, and chicory greens. This process may also be
related to the generation of calcium
oxalate.
An overlooked source of calcium is eggshell, which can be
ground into a powder and mixed into food or a glass of water (Schaafsma et al., 2002). The calcium content of
most foods can be found in the USDA National Nutrient Database.
1.2.3 BONE
HEALTH
Calcium supplementation can have a small effect in improving bone mineral density. This is of
no meaningful benefit to health in children, but in post-menopausal
women the risk of vertebral fractures may be
reduced (Shea et al., 2004).
1.2.4 CARDIOVASCULAR
IMPACT
A study investigating the effects of personal calcium
supplement use on cardiovascular risk in the Women’s Health Initiative Calcium/Vitamin D
Supplementation Study (WHI CaD Study) found a modestly
increased risk of cardiovascular events, particularly myocardial infarction in
postmenopausal women. A broad recommendation of calcium/vitamin D supplements
is therefore not warranted. In contrast,
the authors of a 2013 literature review concluded that the benefits of calcium
supplementation, such as on bone health, appear to outweigh any risk calcium
supplementation may theoretically pose to the cardiovascular health (Downing
and Islam 2013).
1.2.5 HAZARDS AND
TOXICITY
Compared with other metals, the calcium ion and most calcium
compounds have low toxicity. This is not surprising given the very high natural
abundance of calcium compounds in the environment and in organisms. Calcium
poses few serious environmental problems, with kidney stones the most common
side-effect in clinical studies. Acute calcium poisoning is rare, and difficult
to achieve unless calcium compounds are administered intravenously. For
example, the oral median lethal dose (LD50)
for rats for calcium
carbonate and calcium
chloride are 6.45 and 1.4 g/kg, (Lewis, 1996) respectively.
Calcium metal is hazardous because of its sometimes-violent
reactions with water and acids. Calcium metal is found in some drain cleaners,
where it functions to generate heat and calcium
hydroxide that saponifies
the fats and liquefies the proteins (e.g., hair) that block drains. When
swallowed calcium metal has the same effect on the mouth, esophagus and
stomach, and can be fatal (Rumack, 2010).
1.3 HARD WATER
Hard water is water
that has high mineral
content (in contrast with "soft water").
Hard water is formed when water percolates
through deposits of calcium
and magnesium-containing minerals such as limestone, chalk
and dolomite. Hard drinking
water is generally not harmful to one's health, (WHO, 2003)
but can pose serious problems in industrial settings, where water hardness is
monitored to avoid costly breakdowns in boilers,
cooling towers, and other equipment that
handles water. In domestic settings, hard water is often indicated by a lack of
suds
formation when soap is
agitated in water, and by the formation of limescale
in kettles and water heaters. Wherever water hardness is a concern, water softening is commonly used to
reduce hard water's adverse effects.
1.3.1 SOURCES OF
HARDNESS
Water's hardness is determined by the concentration of multivalent cations
in the water. Multivalent cations are cations (positively charged metal complexes) with a charge greater
than 1+. Usually, the cations have the charge of 2+. Common cations found in
hard water include Ca2+ and Mg2+. These ions enter a
water supply by leaching from minerals within an aquifer.
Common calcium-containing minerals are calcite and gypsum.
A common magnesium mineral is dolomite (which also contains calcium). Rainwater and distilled
water are soft, because they contain few ions
(Hermann, 2006).
The following equilibrium reaction describes
the dissolving and formation of calcium carbonate :
CaCO3
(s) + CO2 (aq) + H2O (l) ⇋
Ca2+ (aq) + 2HCO3− (aq)
The reaction can go in either direction. Rain containing
dissolved carbon dioxide can react with calcium carbonate and carry calcium
ions away with it. The calcium carbonate may be re-deposited as calcite as the
carbon dioxide is lost to atmosphere, sometimes forming stalactites and stalagmites.
Calcium and magnesium ions can sometimes be removed by water
softeners
1.3.2 TEMPORARY
HARDNESS
Temporary hardness is a type of water hardness caused by the
presence of dissolved bicarbonate minerals
(calcium bicarbonate and magnesium bicarbonate). When
dissolved, these minerals yield calcium and magnesium cations
(Ca2+, Mg2+) and carbonate and bicarbonate
anions
(CO32-, HCO3-). The presence of the
metal cations makes the water hard. However, unlike the permanent
hardness caused by sulfate
and chloride compounds, this
"temporary" hardness can be reduced either by boiling the water, or
by the addition of lime
(calcium hydroxide) through the
softening process of lime
softening, (Christian et
al., 2005) Boiling promotes the formation of carbonate from the bicarbonate
and precipitates calcium carbonate out of solution, leaving water that is
softer upon cooling.
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