EPA-600/1-77-024
May 1977
Environmental Health Effects Research Series
EFFECTS OF MANGANESE AND
THEIR MODIFICATION BY
HEXAMETAPHOSPHATE
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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EPA-600/1-77-024
May 1977
EFFECTS OF MANGANESE AND THEIR
MODIFICATION BY HEXAMETAPHOSPHATE
by
Richard J. Bull
Water Quality Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects Research Labora-
tory, U. S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute en-
dorsement or recommendation for use.
ii
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FOREWORD
Man and his environment must be protected from the adverse effects of
pesticides, radiation, noise and other forms of pollution, and the unwise
management of solid waste. Efforts to protect the environment require a
focus that recognizes the interplay between the components of our physical
environment—air, water, and land. In Cincinnati, the Environmental
Research Center possesses this multidisciplinary focus through programs
engaged in
studies on the effects of environmental contaminants on
man and the biosphere, and
a search for ways to prevent contamination and to recycle
valuable resources.
The Health Effects Research Laboratory conducts studies to identify
environmental contaminants singly or in combination, discern their re-
lationships, and to detect, define, and quantify their health and economic
effects utilizing appropriate clinical, epidemiological, toxicological,
and socio-economic assessment methodologies.
Source waters for public drinking water supplies occasionally contain
quite high concentrations of manganese. Although this element is essential
for normal health, inhalation of high concentrations of manganese has been
shown to result in a syndrome quite similar to Parkinson's disease. The
current drinking water standard for manganese was established because of
certain esthetic problems associated with its presence which can be
ameliorated by complexation of manganese with certain polyphosphate
compounds. Using this form of water treatment does not remove manganese
from the water. The present study was undertaken to determine if manganese
in drinking water could produce the changes which occur in Parkinson's
disease and to evaluate the practice of manganese complexation in water
treatment.
R. J. Garner
Di rector
Health Effects Research Laboratory
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ABSTRACT
Charles River strain male rats were exposed to Mn concentrations
of 50, 200, 700, and 2000 mg/liter in their drinking water for 6 months.
A second series of animals were exposed to sodium hexametaphosphate
(NaP03)6 at concentrations of 185, 742, 2957 and 7420 mg/liter. These
stoichiometric concentrations of Mn++ and (NaP03)6 were combined in the
drinking water of a third series of animals.
The primary intent of the study was to determine if Mn"1"1" via the
oral route could produce decreases in dopanrine concentrations of the
corpus striata characteristic of the Parkinson-like disease observed
in manganese workers and to correlate such changes with Mn content of
the tissue. Mn++ failed to produce a decrease in corpus striatal dopamine
content. However, a paradoxical decrease in corpus striatal Mn concen-
tration was observed at 200 and 700 mg Mn++/liter of drinking water.
At 2000 mg Mn"1"1"/liter a tendency for corpus striatal Mn to increase
was observed, although they did not exceed control levels even at this
dosage level. Drinking water containing (NaP03)6 resulted in a dose
related decrease in corpus striatal Mn concentrations. Mn++ and (NaP03)6
combinations resulted in a smaller decrease in corpus striatal Mn,
although there was no clear evidence of a trend with dosage in the data.
Both Mn++ and (NaP03)6 alone produced decreased body weight gains
which appeared associated with decreased appetite at high concentrations.
Lower concentrations of Mn++ (50 mg/liter) resulted in an enhanced rate
of early growth. Stoichiometric combinations of Mn++ and (NaP03)6
prevented the decreased rate of growth produced by each agent alone.
We conclude that Mn++ in drinking water presents little danger in
terms of central nervous system involvement in a homogeneous population.
We cannot, however, exclude the involvement of genetic factors which
have been suggested to account for the sporadic occurrence of Parkinson-
like symptoms in manganese workers. The use of (NaP03)6 for Mn complexa-
tion as an alternative to removal in drinking water treatment may be
feasible if carefully controlled to avoid excessive amounts.
IV
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CONTENTS
Foreword i i i
Abstract iv
Figures, Tables vi
Acknowledgments vii
I. Introduction 1
II. Conclusions 2
III. Recommendations 4
IV. Implications for Water Supply Practice 5
V. Methods 8
VI. Results 10
VII. Discussion 18
References 21
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FIGURES
Number
1.
2.
3.
4.
5.
Manganese content of the corpus striatum of rats
exposed to manganese and/or sodium hexameta-
phosphate.
Effects of manganese on growth of weanling rats.
Depression of body weight gain by sodium hexameta-
phosphate in drinking water.
Effects of stoichiometric combinations of manganese
and sodium hexametaphosphate on body weight gain
in weanling rats.
Summary of treatment effects in final body weights.
12
13
14
15
16
TABLES
Number
I.
II.
Page
II
Concentrations of Mn and Sodium Hexametaphosphate
in the Drinking Water of Each Experimental Group. 8
Corpus Striatal Dopamine Concentrations in Rats
Exposed to Manganese and/or Sodium Hexametaphosphate
for 6 Months. 11
vi
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ACKNOWLEDGMENTS
The technical assistance of Mr. S. D. Lutkenhoff, Ms. R. Weister,
and Ms. K. Fisher was excellent and much appreciated.
Ms. J. Roe is thanked for her valuable assistance in preparing this
report.
vii
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SECTION I
INTRODUCTION
Toxicity as a result of excessive exposures to manganese has been
observed in occupational settings. A syndrome clinically indistinguishable
from Parkinson's disease has been reported in mining operations (Mena,
et al., 1967), processing of manganese ore (Cook, et al., 1974) and in
ferromanganese alloy production (Tanaka & Lieben, 1969: Jonderko, et al.,
1971; Smyth, et al., 1973). As in Parkinson's disease, chronic manganism
results in substantial depletion of brain dopamine concentrations (Ehringer
and Hornykiewicz, 1960; Mustafa and Chandra, 1971) and often responds to
treatment with the dopamine precursor L-dihydroxyphenylalamine (Cotzias,
et al., 1971; Goldman, 1972).
Manganese has also been shown to be an essential element (Cotzias,
1958). Although frank deficiency in humans has never been reported, the
concentration of the metal in the modern diet has probably been reduced
by food processing (Schroeder, et al., 1966).
The USEPA drinking water standard for manganese has been established
for esthetic reasons at 0.05 ing/liter. Esthetic objectives to manganese
in public water supplies may be controlled by complexation with various
polyphosphate compounds. It could be argued that if the esthetic require-
ments of the drinking water standards can be safely managed by complexation,
an elevated manganese concentration in drinking water could be tolerated
or may even be desirable.
A recent paper (Bonilla & Diez-Ewald, 1974) indicated that brain
dopamine levels were reduced by about 50% in rats drinking water containing
5 mg MnCl2 per ml for 7 months. The present study was undertaken to develop
dose-response information on this effect of manganese and to determine if
complexation with sodium hexametaphosphate modified the effect in any way.
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SECTION II
CONCLUSIONS
The drinking water standard for Mn currently in force was established
for esthetic reasons. Knowledge of this fact has led certain water
treatment facilities to employ complexation of Mn with polyphosphate
compounds as an alternative to Mn removal from raw water. This does
satisfy esthetic objections to Mn in drinking water. However, a question
remains as to what limit should be placed upon Mn concentrations in drink-
ing water from a health standpoint. Secondary questions arise as to the
toxic properties of the complexing agent and the complex itself.
Certain tentative conclusions may be arrived at on the basis of the
present study. The most severe toxic effect of chronic manganism involves
the production of a syndrome which is clinically indistinguishable from
Parkinson's disease. In human cases of the disease, this syndrome has
been shown to be associated with substantial decreases in brain dopamine
content. Similarly, injections of Mn, and in one study, oral ingestion
of Mn++ have been shown to produce a decrease in brain dopamine concentra-
tions. We observe no such decrease in our experimental animals with
exposures of up to 2000 mg Mn++/liter for 6 months. Therefore, we can
conclude that orally administered Mn++ does not appear a substantial
hazard in this regard in a homogeneous population such as our test animals.
We cannot, however, exclude the possibility, mentioned in the literature,
that a substantial minority of the human population may be susceptible to
lower concentrations of Mn on a genetic basis. Our work indicated a
dynamic regulation of tissue Mn concentrations in the face of widely
differing environmental exposures. A defect in this regulation produced
by either environmental or genetic factors could dramatically increase
sensitivity to Mn++.
The present study did not reveal any overwhelming reasons for
precluding the practice of Mn++ complexation in treatment of'drinking
water. The results do suggest that care should be exercised in the use
of polyphosphates for this and other purposes. At concentrations of 742
mg (NaP03)6 and above frank depletion of tissue Mn was evident. Undoubted-
ly, the absorption of other trace nutrients were similarly affected. Trends
in the data presented on growth of the animals suggested that concentrations
lower than 742 mg (NaP03)6 may have some impact on health over the long
term. Mn++ and (NaP03)6 mutually antagonized one another's effects on
body weight gains. Consequently, the practice of complexation by polyphos-
phates has some merit if the water treatment dosage is carefully titrated
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to avoid addition to excessive polyphosphate. Some note should be added
to limit this practice to realistic concentrations of manganese, perhaps
1-2 nig/liter. Even in combination there were some indications of depleted
tissue Mn concentrations. Additionally, the mutual antagonism of the two
compounds broke down, perhaps due to the toxicity of the complex, at their
highest concentrations in combination.
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SECTION III
RECOMMENDATIONS
1. The present drinking water standard for manganese should be maintained
at 0.05 nig/liter for esthetic reasons.
2. Complexation of manganese present in raw water by sodium hexametaphos-
phate be accepted as conforming to the above standard as long as the
treatment plant confirms on a routine basis that the amount of sodium
hexametaphosphate does not exceed the requirement by more than 10% and
that manganese concentrations being treated do not exceed 1 mg/liter.
3. The effects of manganese on neonatal animals requires further study.
Some limited evidence indicates that mothers subjected to manganese
loading excrete large amounts of manganese in their milk. It is
possible that less developed mechanisms for controlling absorption,
distribution and excretion of manganese in younger animals may result
in adverse effects at much lower concentrations. Because of the asso-
ciation of chronic manganism with effects on the control nervous
system any studies initiated in this area should emphasize behavioral,
neurochemical and neurophysiological parameters.
4. Polyphosphate compounds commonly used in water treatment should also
be evaluated experimentally. Data derived wholly from a single com-
pound, sodium hexametaphosphate, is clearly too little with which to
extrapolate to an entire class of compounds. It is possible that
certain of these compounds could enhance absorption of the offending
metal or that the complex itself may possess unique toxicological
properties.
5. The biological behavior of other metal complexes with polyphosphate
compounds should also be studied. Polyphosphate compounds are
apparently used occasionally when iron is present in raw water at
concentrations in excess of the drinking water standard. Behavior
of a manganese complex does not necessarily extrapolate to other
metals. The possibility that polyphosphates may enhance the leaching
of toxic metals from the distribution system and/or increase their
bioavailability should also be entertained.
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SECTION IV
IMPLICATIONS FOR WATER SUPPLY PRACTICE
One of the basic reasons for undertaking the present study was to
examine the practice of utilizing polyphosphate compounds for water
treatment. This treatment is generally applied to meet certain formal
or informal esthetic requirements for drinking water. In contrast to
the necessity for actual removal of chemicals which are known for their
potential as health hazards, this method of treatment is symptomatic and
does not involve removal of the offending agent. Rather a complex is
formed which remains in the drinking water. Thus one is faced with a
three-fold problem when considering possible hazards to the health of
populations consuming water treated in such a manner. First, some
hazard may be associated with the offending chemical. Second, the hazard
associated with the treatment chemical must be evaluated. Third, the
complex formed may have distinct toxicological problems of its own or
the toxicity of one or both of the parent compounds may be altered by
changes in absorption, distribution or excretion.
Before this type of treatment is even contemplated it must be
established that the offending chemical is relatively non-toxic. Treat-
ment failure or improper treatment would be too great a risk to assume
on a daily basis. For example, such treatment would have to be ruled
out a priori for such toxic metals as Pb, Cd, and Hg. The present study
and prior studies have indicated that manganese fits into the classifi-
cation of a chemical with relatively low toxicity via the oral route,
although certain reservations must be acknowledged in terms of manganese
toxicity in young animals (see recommendations). The polyphosphate
compounds ordinarily used in water treatment also have been regarded as
being relatively safe chemicals in terms of human health. The present
study bears this out to a large extent. However, evidence was obtained
that high levels of polyphosphates interfered with essential trace metal
metabolism. More alarming was a trend that developed in the same direc-
tion at lower concentrations as well, even though the individual results
were not statistically significant. The fact that addition of stoichio-
metric concentrations of manganese to the drinking water resulted in a
significant enhancement of growth rate at 742 mg (NaPOaJg/liter suggests
that this concentration does interfere with trace metal metabolism.
Such an effect may have longer-term nutritional consequences in a human
population where individual diets vary quite widely. Relatively small
concentrations of polyphosphates in the drinking water may be sufficient
to induce deficiencies in individuals consuming a diet marginal in
essential trace metals.
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Since both manganese and sodium hexametaphosphate do produce some signs
of toxicity at high concentrations, it is necessary that some limits be
established for manganese complexation as a water treatment practice. Both
manganese and sodium hexametaphosphate alone appeared more toxic than the
complex of the two. Consequently, if the treatment is used it is important
that use of sodium hexametaphosphate be carefully adjusted to the require-
ment posed by manganese in the source water. Barring a source water which
varies widely and unpredictably, a water treatment plant with routine analyt-
ical capabilities for trace metal analysis should have little difficulty in
maintaining a sodium hexametaphosphate dosage rate which does not exceed the
stoichiometric manganese content by more than 10%. Manganese at a concentra-
tion of 700 mg/liter did not depress growth relative to control animals.
However, since lower concentrations resulted in an enhancement of early
growth and addition of a stoichiometric quantity of (NaP03)6 to water con-
taining 700 mg Mn++/liter significantly enhanced growth, it is apparent that
this concentration of manganese was beyond the optimum manganese intake.
Even though no long term effects appears to result from this level, the
pattern dictates a conservative view until the effects of manganese loading
can be more fully established in neonatal animals. Taking, therefore, a
concentration of 200 mg Mn++/liter as a "no effect" level, use of polyphos-
phate treatment for concentrations of manganese up to 1 mg/liter in raw
water should present little hazard to health. This would not more than
double the average manganese present in the human diet assuming a 2 liter
daily consumption. A safety factor of 200 would then apply in terms of
health even in the event of complete treatment failure. Similarly, if one
operates within the above stated restriction of not exceeding the require-
ment for sodium hexametaphosphate by 10% not more than 4 mg (NaP03)6/liter
would be added leaving an excess of 0.4 mg of non-complexed (NaP03)6/liter.
Concentrations of 185 mg (NaP03)6/liter would without significant effect on
tissue manganese content and growth. This provides a safety factor for
non-complexed (NaP03)6 of 462. If no metals were present to complex (NaP03)6
the safety factor would be reduced to 46 in the long-term. Short-term
overdosage should present little hazard. The safety factor for the Mn++
plus (NaP03)6 complex would be 700 on the basis of present experiments.
It should be noted that these safety factors have been calculated in
terms of concentration rather than dose/unit body weight as. is ordinarily
done. Interactions between polyphosphates and trace metals appear to
involve chemical interactions within the gastrointestinal tract and would
be expected to be more closely related to concentration than to dose/unit
body weight expected of a systemic physiological interaction between the two
chemicals. However, since man consumes less water per unit of body weight
than rats, the safety factors in terms of dose/unit body weight would be
somewhat greater than indicated above.
In conclusion, sodium hexametaphosphate, and possibly other polyphos-
phafce compounds, can be used without undue risk to satisfy esthetic standards
for manganese in drinking water. The use of polyphosphates-for the purpose
of removing esthetic objections arising from the presence of metal ions,
however, must be governed by the amount of the offending metal present.
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Excess polyphosphate may prevent absorption of essential trace metals from
the diet and thus cannot be considered completely inert from a public health
standpoint. Consequently, concentrations of polyphosphate greatly in excess
of a metal ion defined requirement should be avoided as a questionable
public health practice that may have long-term nutritional repercussions.
Therefore, a recommendation is made that the practice of complexing.
manganese to meet the EPA drinking water standard be restricted to concen-
trations of Mn which do not exceed 1 mg/liter. In addition, the dosage
rate for sodium hexametaphosphate should not exceed the metal ion require-
ment by more than 10% as defined by routine analysis of the raw water.
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SECTION V
METHODS
Male, weanling Charles River Strain rats (Sprague-Dawley) were
distributed into 12 experimental groups of 9 animals each. The drinking
water (distilled) contained either MnCl2, MnCl2 in stoichiometric
combination with sodium hexametaphosphate ((NaP03)6), or (NaP03)6 alone
as shown in Table 1.
TABLE 1. CONCENTRATIONS OF Mn++ and/or (NaP03)6 UTILIZED
Mn
Mn++ + (NaP03)6
(NaP03),
Mn++ 50 mg/ liter
(NaP03)6
Mn++ 200
(NaP03)6
Mn++ 700
(NaP03)6
Mn++ 2000
(NaP03)6
50 mg/ liter
185 mg/ liter 185 mg/ liter
200
742 742
700
2957 2957
2000
7420 7420
A control group of 18 animals was maintained on distilled water. All
animals were allowed free access to commercial laboratory chow (Teklad,
stated Mn4"*" concentration 94 ug/g). Food and water consumption were
monitored and animals were periodically weighed throughout the study.
At the end of 6 months, animals were sacrificed, the brains rapidly
removed and placed in a cold box. The corpus striatum was dissected out
and forzen on a block of dry ice. The pair of striata from each animal
were then weighed and homogenized in 0.1 N perchloric acid (60 volumes).
8
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Extracts were centrifuged at 10,000 X g for 10 min and the supernatant
decanted. 2.0 ml was taken from dopamine analysis and the remainder
was utilized for analysis of manganese content. The samples were pre-
served at -90° C until analysis could be performed. Sacrifice of
animals was scheduled so that one-half of the control animals (9) were
the first and the other half the last animals sacrificed to eliminate
actifactual trends in the analyses.
A radioassay which made use of the 0-methylation reaction of
catechol-0-methyl transferase with S-adenosylmethionine (methyl-3H)
(6.8-12.6 Cj/mM) was utilized for dopamine analysis. The procedure was
a modification of that described by Coyle and Henry (1973).
Manganese determinations were made directly following neutralization
and removal of perchloric acid by K2C03 by non-flame atomic absorption
spectroscopy using a Varian AA-6 fitted with a model 63 carbon rod atomizer.
Calculations were made from standard curves obtained from known concentra-
tions of manganese dissolved in 0.1 N perchloric acid. Tissue analysis of
both manganese and dopamine were performed blind. The data was tested for
significance by analysis of variance and student's t-test.
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SECTION VI
RESULTS
The condition of all animals in the study was generally good. No
evidence of gross neurological damage was apparent in any experimental group.
The levels of dopamine in the corpus striata of rats on the study did
not vary systematically with dose of either Mn or (NaP03)6 (Table II).
Corpus striatal dopamine concentrations did exhibit differences which were
statistically different from control in certain groups. The maximum
deviations were an apparent 17% decrease of dopamine in the 742 mg (NaP03)e/
liter group and an apparent 13% increase in the group receiving 2000 mg Mn /
liter. These changes were .not dose-related and are small enough to be of
questionable biological significance.
Manganese content of the corpus s|riatum exhibited a paradoxical
decrease with increasing intake of Mn (Fig. 1). This amounted to a 50%
decrease at 200 mg Mn++/liter of drinking water. A gradual increase of
corpus striatal manganese content, towards control levels, was observed at
higher concentrations of manganese.
Corpus striatal manganese was also found to decrease with increasing
concentrations of (NaP03)6 in the drinking water (Fig. 1). The decrease was
dose related, but appeared to be maximal at 2947 mg (NaPOaJe/liter. Chemi-
cally equivalent concentrations of Mn++ and (NaP03)6 resulted in an average
decrease in corpus striatal manganese of about 30%. However, there appeared
to be no trend with dose. There was no significant correlation between the
individual concentrations of manganese and dopamine in corpus striata
(r = 0.034).
Animals on the different treatments did exhibit significant differences
in growth and final body weights relative to control animals (Fig. 2).
Inclusion of 50 mg MITY liter of drinking water resulted in increased body
weights. Although not statistically significant at termination of the study,
the difference apparently resulted from a small but significant enhancement
of early growth. At the start of the experiment this group averaged
57.1 ± 1.6 g compared to 55.0 ± 1.9 (SEM) for the control group. After 51
days on the study the body weights of these groups were 438 ± 9 and 412 ± 9,
respectively (P < 0.05). At intermediate concentrations of manganese this
difference from the control group were not apparent. Manganese at a
concentration of 2000 mg/liter resulted in a significant depression in
growth rate, which was already apparent 3 weeks into the study. Despite
10
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the early appearance of growth inhibition only a 10% decrement was observed
from control at the end of 6 months and no deaths were observed.
TABLE 2. CORPUS STRIATAL DOPAMINE
CONCENTRATIONS IN RATS EXPOSED TO MANGANESE
AND/OR SODIUM HEXAMETAPHOSPHATE FOR 6 MONTHS
(NaP03)6
mg/1
0
185
742
2957
7420
Mn++, mg/1
0 50 200
11.2 ± 0.4* 10.0 ± 0.3 11.1 ± 0.4
11.3 ± 0.3 10.7 ± 0.6
9.3 ±0.4 - 9.8 ± 0.4
12.5 ±0.5
11.8 ±0.4
700 2000
10.7 ± 0.5 12.8 ± 0.3
-
-
12.1 ± 0.3
10.6 ± 0.3
dopamine/g tissue ± S.E.M.
Sodium hexametaphosphate (NaP03)6 was also observed to depress body
weight at high concentrations (Fig 3). Unlike manganese, effects on body
weight did not become apparent for two months even at 7420 mg (NaP03)6/liter.
Effects of lower concentrations of (NaP03)6 on body weight appeared to be
characterized by later departures from the control growth curve. This trend
was observed at all concentrations, suggesting that continued exposure may
have resulted in significant differences from control at concentrations below
2957 mg/liter, the lowest dose to show significant differences in the
present study.
Stoichiometric combinations of Mn++ and (NaP03)6 indicated that the two
substances were basically antagonistic in their effects upon growth rate
(Fig 4). The combination of 700 mg Mn++ and 2957 mg (NaP03)6/liter resulted
in an enhancement of body weight gain relative to control at 6 months
(P < 0.05). The latency of this effect resembled that of (NaP03)6 alone as
opposed to Mn++ alone; not becoming evident until the end of the second month
of the study. Combination of 2000 mg Mn"1"* with 7420 mg (NaP03)6/liter of
drinking water resulted in a small depression of body weight at 6 months.
Although not significantly different from the control group (0.05 < P < 0.1),
it 'did represent a substantial depression when compared with the group
receiving 700 mg Mn++ and 2940 mg (NaP03)6/liter (P < 0.01). A summary of
final body weights for each experimental group is provided in Fig 5.
11
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VI
in
1.0
E
3
in
3
Q.
l_
o
U
c
O)
*-
c
o
o
•t-
0.5
I
O (NoP03)6
-i- (NoP03)g
50
I
185
200
700
2000
Manganese, mg/l
742
2957
I
7420
Sodium hexametaphosphate, mg/l
Figure 1. Effects of treatments upon the manganese
content of the rate corpus striatum at 6 months
of exposure. Horizonal lines indicate the average
(solid) ± SEM (dashed) of the control group. All
other points show the average ± of the indicated
treatment group.
12
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90
Do,.
Figure 2. Effects of manganese in drinking water
on growth of weanling rats. The 200 mg Mn*+/liter
group was omitted because it coincided with the
control group.
'85 mg/t
200 mg.'l * (McPQ^Ig, 742 mg
700 mq.'i • I NoPOjlg, Z%57 rr.
2000 ™g/ i - lNDP05)6, 7420
120 150 180
Doys
IN0P03J6, IBS «,,/!
(N0P03>6. 742 ™9/l
iNoPO.) . 2957 mq/l
— (NjPO.L . 7420 mg/l
90
Days
Figure 3. Depression of body weight
gain by weanling rats produced by
sodium hexametaphosphate in drinking
water.
Figure 4. Effects of stoichiometric combinations of Mn++ and (NaP03)6 on body weight
gain of weanling rats.
-------
700
o>
J
O
CO
650
600
50
200
700
20OO
Manganese, mg/l
185 742 2957 7420
Sodium hexametaphosphate, mg/l
Figure 5. Summary of treatment effects on final body
weights. Solid horizontal line represents the average
weight of 18 control animals with dashed lines showing
the SEM. All other points show the average ± SEM of
the indicated treatment group.
14
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Depressions in growth produced by both Mn++ and (NaP03)6 were associ-
ated with decreases in food consumption (data not shown). The latency of
decreases in food consumption were very closely associated with the periods
in which differences in body weight were first noted. A 10% decrease in the
consumption of chow was observed with 2000 mg Mn+^/liter during the third
week of the study whereas consumption of food by the 7420 mg (NaP03)6/liter
group was 4% above control. During the sixth week, however, the latter
group's consumption of laboratory chow decreased by 10% relative to controls
and remained at this level through the twelfth week of the study. Beyond
this point, food consumption by this group was consistently below controls
but only by 3-5%. Conversely, groups which experienced better weight gains
than controls evidenced significantly higher food consumpton which coincided
in latency with divergence of body weights from controls. Animals drinking
water containing 50 mg Mn++/liter consumed 6.1% more chow than controls for
the first 4 weeks of the study. This difference had disappeared by the
eighth week. Drinking water containing 700 mg Mn++ and 2952 mg (NaP03)6/
liter exhibited a 5% increase in food consumption during weeks 10-20. The
increased growth rate of this group became evident that the day 80 weighing.
15
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SECTION VII
DISCUSSION
The principle purpose of this study was to determine if oral manganese
exposures might result in neurological damage similar to that observed in
occupational settings which primarily involve inhalation exposures (Mena,
et al.5 1967; Cook, et al., 1974; Tanaka & Lieben, 1969; Jonerko, et al.,
1971; Smyth, et al,, 1973). The data reported here indicates that substan-
tial intake of Mn (up to 100-200 mg/Kg per day for a 6 month period) do
not produce the characteristic decrease in dopamine associated with chronic
manganism in the male rat.' The few differences observed between experimental
groups appeared to result from random variation. These data support the
conclusion that the drinking water standard for manganese (0.05 mg/liter) is
more than adequate to protect against this effect of manganese. The results
are different from those of Bonilla and Diez-Ewald (1974). Their study
utilized a single exposure level, equivalent to 2182 mg Mn++/liter, for a 7
month period. These authors employed female rats, which might explain the
difference in results. Some caution must be exercised to avoid overinter-
pretation of our negative results, however. Occurence of chronic manganism
in exposed populations is not simply a function of concentration. The fact
that the disease often develops within a few years in certain individuals
but never occurs in other individuals working under the same conditions for
much longer periods has led to a suspicion that other factors, such as
nutritional status (Mena et al., 1969) or genetic factors (Cotzias, et al.,
1972), are necessary determinants of the disease. Consequently, studies in
genetically homogeneous animals with nutritionally adequate diets may not
completely define the risk. In addition, manganese loads are known to
produce changes in carbohydrate and lipid metabolism which could have
implications in chronic disease states outside the scope of the present
study.
The various treatments did impact on the corpus striatal manganese
concentrations. Most interesting was the paradoxical decrease in the
tissue's manganese content at 200 mg Mn /liter of drinking water. While
there is no specifically stated precedent for a decrease with increased
exposures, some data appears in a paper from Cotzias1 laboratory (Hughes
et al., 1966) indicating a similar decrease of manganese concentrations in
the diaphragm of mice exposed to 20 mg Mn++/liter in a milk diet for 54 days.
Excretion of manganese is known to be very sensitive to manganese load, both
in humans (Borg and Cotzias, 1958) and experimental animal-s (Britton and
Cotzias, 1966). Distribution of manganese can be influenced by adrenocorti-
cal hormones (Hughes et al., 1966) and is known to redistribute with loading
16
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(Papavasiliou et al., 1966). To explain an actual decrease in a tissue
concentration in the face of increased exposure to the metal would require
active regulation and could not be explained merely by increased excretion
due to mass action. Obviously, this potentially important result must be
confirmed and coupled with more extensive documentation of manganese distri-
bution with varying manganese loads. Nevertheless, it is apparent that
corpus striatal content of manganese is regulated within very close limits
and even extreme changes in oral intake have relatively small impacts on
the actual tissue concentrations. The tendency for corpus striatal mangan-
ese to gradually increase with concentrations above 200 mg Mn++/liter
suggests that higher concentrations may have resulted in actual elevations
in the tissue's content of the metal. Elevations of whole brain manganese
concentrations have been documented in lambs receiving 4030 ppm Mn in the
diet (Watson et al., 1973).
The dose-related depletion of corpus striatal manganese with (NaP03)e
may be presumably explained by the propensity of this chemical to complex
manganese making it less available for absorption. High phosphate to
calcium ratios are known to limit the absorption of manganese (Underwood,
1971). The effect of stoichiometric combinations of Mn++ and (NaP03)6 on
corpus striatal manganese concentrations are not so easily understood.
Although the curve obtained with the concentration of the combination tends
to possess some of the characteristics of that observed with Mn++ alone,
the trends are less clear. Possibly interactions of Mn++ and (NaP03)6 with
the metabolism of other essential elements may be involved (Mena, et al.,
1969; Watson, et al., 1973).
Interactions of Mn++ and (NaP03)6 were better defined by the effects of
the differing treatments of growth. In all cases, the effects of the treat-
ments appeared to exerted through changes in appetite rather than direct
toxicity of the chemicals. The optimum intake of manganese varies signi-
ficantly depending upon the parameter being studied (Underwood, 1971).
Holtkamp and Hill (1950) indicated that increasing manganese intake to
2 mg/day was optimal for growth of rats around 30 days of age. Rats on the
present study consumed an average of 17 g of chow per day during this
period. At 94 ug Mn/g, the manganese in the diet amounted to 1.6 mg/rat per
day. Consequently, it is reasonable to conclude that the increased rate of
body weight gain in animals consuming 50 mg Mn /liter in their drinking
water resulted from the nutritional contribution. The appetite depressant
effects of 2000 mg Mn /liter and the associated decreases in body weight
gain are consistent with the results of previous studies as well (Underwood,
1971). Sodium hexametaphosphate, in contrast to Mn++, possessed only a
depressant effect on body weight.
The slightly less than optimal manganese for growth present in the
laboratory chow resulted in interactions between Mn and (NaP03)6 becoming
evident. Most conspicuous was the shifting of the optimum growth from 50
to 700 mg Mn /liter by addition of stoichiometric amounts of (NaP03)6.
17
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This finding suggests that the biological availability of the (NaP03)g
complex of Mn++ is less than 10% of that of HnCl2. Conversely, modification
of the effects of (NaP03)5 on body weight gain by manganese indicates that
the toxicity of this material is probably exerted primarily through complex-
ation of essential metals. Absorption of manganese, itself, has long been
known to be inhibited by lowering of the Ca:P ratio in the diet (Wachtel,
et al., 1943). However, it cannot be concluded that Mn"1"1" compilation alone
is responsible for the effects of (NaP03)6 in that other essential metals,
such as Fe, Mg, and Ca, are likely to be affected as well. Complexation of
these metals by (NaP03)6 would also be decreased by stoichiometric addition
of Mn to (NaP03)6 solutions particularly at the concentrations utilized
in the present study. As a result of the differing latency of growth
enhancement by the 700 rag Mn++ and 2957 mg (NaP03)6/liter combination rela-
tive to that of 50 mg Mn /liter alone, a more general effect of (NaP03)6
than a specific interaction with Mn++ seems indicated.
18
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REFERENCES
Bonilla, E. and Diez-Ewald, M. (1974) Effect of L-dopa on brain concentra-
tions of dopamine and homovanillic acid in rats after chronic manganese
chloride administration. J. Neurochem. 22, 297-99.
Borg, D. C. and Cotzias, G. C. (1958) Manganese metabolism in man: Rapid
exchange of Mn56 with tissue as demonstrated by blood clearance and liver
uptake. J. Clin. Invest. 3tf, 1269-78.
Britton, A. A. and Cotzias, G. C. (1966) Dependence of manganese turnover
on intake. Am. J. Physio!.- 211, 203-6.
Cook, D. G., Fahn, S. and Brait, K. A. (1974) Chronic manganese intoxica-
tion. Arch. Neurol. 30, 59-64.
Cotzias, G. C. (1958) Manganese in health and disease. Physiol. Rev.
38, 503-32.
Cotzias, G. C., Papavasiliou, P. S., Ginos, J., Steck, A. and Duby, S. (1971)
Metabolic modification of Parkinson's disease and of chronic manganese
poisoning. Ann. Rev. Med. 22, 305-26.
Cotzias, G. C., Tang, L. C., Miller, S. T., Sladic-Simic, D. and Hurley,
L. S. (1972) A mutation influencing the transportation of manganese,
L-dopa and L-tryptophan. Science 176, 410-2.
Coyle, J. T. and Henry, D. (1973) Catecholamines in fetal and newborn rat
brain. J. Neurochem. 21, 61-7.
Ehringer, H. and Hornykiewicz, 0. (1960) Verteilung von Noradrenalin
und Dopamin (3-Hydrory tyramin) im gehren des Menshen und ihr Verhalten
bein Erkrankungen des Extrapyramidalen Systems. Klin. Wshr. 38_, 1236-
Goldman, M. E. (1972) Levo-dihydroxyphenylalanine - Parkinson's disease
and manganese poisoning. Industrial Med. 41_, 12-5.
Holtkamp, D. S. and Hill, R. M. (1950) The effect on growth of the level
of manganese in the diet of rats, with some observations on the manganese-
thiamine relationship. J. Nutr. 4]_, 307-16.
19
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Hughes, E. R., Miller, S. T. and Cotzias, G. C. (1966) Tissue concentra-
tions of manganese and adrenal function. Am. J. Physiol. 211, 207-10.
Oonderko, G., Kujawska, A. and Langauer-Lewowicka, H. (1971) Problems
of chronic manganese poisoning on the basis of investigations of workers
at a manganese alloy foundry. Int. Arch. Arbeitsmed. 28, 250-64,
Mena, I., Horiuchi, K., Burke, K., and Cotzias, G. C. (1969) Chronic
manganese poisoning. Individual susceptibility and absorption of iron.
Neurol. J9, 1000-6.
Mena, I., Marin, 0., Fuenzalida, S. and Cotzias, G. C. (1967) Chronic
manganese poisoning. Clinical picture and manganese turnover. Neurol.
17., 128-36.
Mustafa, S. J. and Chandra, S. V. (1971) Levels of 5-hydroxytyryptamine,
dopamine, and norepinephrine in whole brain of rabbits in chronic manganese
toxicity. J. Neurochetn. 1£, 931-3.
Papavasiliou, P. S., Miller, S. T. and Cotzias, G. C. (1966) Role of
liver in regulating distribution and excretion of manganese. Am. J.
Physiol. 211, 211-6.
Schroeder, H. A., Balassa, J. J. and Tipton, J. H. (1966) Essential trace
metals in man: Manganese, a study in homeostasis. J. Chron. Pis. 19,
545-71.
Smyth, L. T., Ruhf, R. C., Whitman, N. E. and Dugan, T. (1973) Clinical
manganism and exposure to manganese in the production and processing of
ferromanganese alloy. J. Occup. Med. 15, 101-9.
Tanaka, S. and Lieben, J. (1969) Manganese poisoning and exposure in
Pennsylvania. Arch. Environ. Health 19, 674-84.
Underwood, E. J. (1971) Trace elements in human and animal nutrition
3rd ed. Academic Press, N. Y. pp. 177-207.
Wachtel, L. W., Elvehjem, C. A. and Hart, E. B. (1943) Studies on the
physiology of manganese in the rat. Am. J. Physiol. 140, 72-82.
Watson, L. T., Ammermun, C. B., Feaster, J. B. and Roessler, C. E. (1973)
Influence of manganese intake on metabolism of manganese and other minerals
in sheep. J. Animal Sci. 36, 131-6.
20
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-77-021*
3. RECIPIENT'S ACCESSIOWNO.
4. TITLE AND SUBTITLE
Effects of Manganese and their Modification by
Hexametaphosphate
5. REPORT DATE
May 1977 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
'(Uchard J. Bull
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Health Effects Research Laboratory - Gin., OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 1*5268
10. PROGRAM ELEMENT NO.
1CC614
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Same as above
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
RACT • .1
The ability of oral MnTT to produce the depletions of dopamine in the corpus
striata characteristic of the Parkinson-like syndrome in manganese workers was
examined in rats. Mn++ failed to affect dopamine levels at concentrations of up to
2000 mg/liter of drinking water and six months of continuous exposure.
A second objective ot this work was to study the biological interations
between Mn++ and sodium hexametaphosphate [(NaPO-),-] administered simultaneously in
drinking water. Some evidence was obtained that-^hlgh concentrations of (NaPO_)fi
depleted tissue Mn++ and produced decreased body weight gains over the experimental
period. Similarly, Mn++ in high concentrations resulted in decreased growth of
weanling animals. Stoichiometric combinations of Mh+t and (NaPCL)g prevented the
decreased rate of growth produced by either agent alone. ->
On the basis of these results, we conclude that Mi*"1" in drinking water presents
little hazard in terms of central nervous system involvement in a homogeneous
population. We cannot, however, exclude the involvement of genetic factors which
have been suggested to account for the sporadic occurrence of Parkinson-like
symptoms in Mn++workers. The use of (NaPCL)g for MI++ complexation as an alter-
native to removal in drinking water treatmenB appears safe from a health standpoint
if carefully controlled to avoid excessive amounts of (NaPO-)/-.
3 o
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Manganese
Drinking water
Dopamine
Toxicity
Rats
Potable water
sodium-hexametaphosphate
corpus striata,
Parkinson's disease
13B
8. DISTRIBUTION STATEMENT
Release to Public ,
19. SECURITY CLASS (ThisReport)
TTnr>1
21. NO. OF PAGES
29
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
21
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