The Science of the Total Environment 302 (2003) 127–144

Epidemiological investigation on chronic copper toxicity to

children exposed via the public drinking water supply
¨ P. Zietza,*, Hermann H. Dieterb, Max Lakomekc, Heide Schneidera, Barabara Keßler-
Bjorn
Gaedtkea, Hartmut Dunkelberga
a
Medical Institute of General Hygiene and Environmental Health, University of Gottingen,
¨ Windausweg 2, D-37073 Gottingen,
¨
Germany
b
Department for Water-, Soil and Air Hygiene, Federal Environmental Agency, Corrensplatz 1, D-14195 Berlin, Germany
c
¨
Department of Pediatrics, University of Gottingen, ¨
Robert-Koch-Straße 40, D-37075 Gottingen, Germany

Received 2 July 2002; accepted 11 September 2002

Abstract

Copper in drinking water has been associated with Non-Indian Childhood Cirrhosis (NICC), a form of early
childhood liver cirrhosis. This epidemiological study examines the exposition of infants to increased copper
concentrations through drinking water from public water supplies in Berlin, Germany, and if this dietary copper
intake can cause liver damage in early childhood. In total, water samples from 2944 households with infants were
tested for copper. Mean copper concentrations in the two different types of collected composite samples were 0.44
and 0.56 mgyl, respectively. Families having a copper concentration at or above 0.8 mgyl in one or both of the
composite samples (29.9% of all sampled households) and a defined minimum ingestion of tap water of their infant
were recommended to undergo a paediatric examination. Nearly every of the 541 recommended infants were examined
by a local paediatrician and of these 183 received a blood serum analysis, too. None of the infants had clear signs of
a liver disease although a few serum parameters lay outside the accompanying reference range and abdominal
ultrasound imaging gave slightly unusual results in five cases. Additionally, no signs of a negative health effect could
be found in the statistical analysis of the serum parameters GOT, GPT, GGT, total bilirubin, serum copper, or
ceruloplasmin in relation to estimated daily and total copper intakes of the infants from tap water. No dose relation
of serum parameters and estimated copper intakes could be established. From the results of the study, no confirmed
indication of a liver malfunction in infants whose food had been prepared using tap water with an elevated copper
concentration could be found and, therefore, no indication of a hazard due to copper pipes connected to public water
supplies could be detected.
䊚 2002 Elsevier Science B.V. All rights reserved.

Keywords: Copper; Heavy metals; Non-Indian Childhood Cirrhosis, NICC; Public water supply; Tap water

*Corresponding author. Tel.: q49-551-394971; fax: q49-551-394957.

E-mail address: bzietz@gwdg.de (B.P. Zietz).

0048-9697/03/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 8 - 9 6 9 7 Ž 0 2 . 0 0 3 9 9 - 6
128 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
1. Introduction
Copper is an essential trace element in the
human body, and is an indispensable component
of many enzyme systems. But a number of path-
ogenic characteristics are attributed to this element.
The role of copper in connection with a form of
early childhood liver cirrhosis, Non-Indian Child-
hood Cirrhosis (NICC), has been the topic of
much discussion and was investigated in several
studies. This disease is similar to Indian Childhood
Cirrhosis (ICC) which has now been known for
more than a century (first documentation by Sen,
1887) and it occurs mainly in India. It is an early
childhood disease, which accompanies an exces-
sive accumulation of copper in the liver and leads
finally to liver cirrhosis. ICC is ascribed by many
authors to a high intake of copper derived from
milk stored or boiled in copper or brass vessels
(Tanner, 1986, 1998).
In Germany, cases of NICC were documented
from 1979 onwards in areas of the Bavarian Forest
and the Emsland and later in northern Germany
(Dieter et al., 1999; Eife et al., 1991; Schafer
¨ and
¨
Schumann, 1991). The course of the disease and
the histological findings are similar to those of
ICC (Muller-Hocker
¨ ¨ ¨ ¨
et al., 1988; Muller-Hocker,
1999). Previous studies suggest that here copper
has an etiological role too (Eife et al., 1991; Bent
¨
and Bohm, 1995; Eife et al., 1987–1988, 1999;
¨
von Muhlendahl and Lange, 1994; Muller¨ et al.,
1999a; Tanner et al., 1979; Trollmann and Behrens,
1998). The intoxication purportedly originated
through high copper levels in the tap water of
households with copper pipes and through the use
of this water in the preparation of the infants’
food.
In 1993, the World Health Organization pub-
lished a provisional guideline for copper in drink-
ing water: the concentration of copper should not
exceed 2 mgyl (World Health Organization, 1993,
compare with Fitzgerald, 1998). This limit was
also introduced in 1998 on a European basis
(European Union, 1998) and has recently been
transferred into German law (TrinkwV, 2001). A
comparison of all published cases of NICC reveals
that all of the households in question used drinking
water from wells, and that this water did not
correspond to the drinking water guidelines (that
means pH-6.5 in these cases). However, in Ger-
many, approximately 60% of the pipes used in
household plumbing are made of copper today
(Arens, 1999).
The studied area of Berlin is supplied by nine
water supply facilities (Beelitzhof, Friedrichshag-
en, Kaulsdorf, Kladow, Spandau, Stolpe, Tegel,
Tiefwerder, Wuhlheide) that delivered 222.3 mil-
lion m3 in the year 2000 to 250 000 domestic
water connections via a distribution network of
7800 km length (Berliner Wasserbetriebe, 2001).
2. Materials and methods
The aim of the present study was to determine
the extent of excessive copper concentrations in
the tap water of households with copper pipes
connected to the public water supply and to reveal
a possible association between the copper content
of tap water used for baby food and the risk of
early childhood liver disease. The design of this
investigation is a cross-sectional study including
paediatric examinations of infants with elevated
copper intake through tap water and follow-up of
subjects with unclear examination results. The
target group of the investigation comprises infants
up to the age of 18 months living in households
with copper plumbing. The study was approved
by the ethics committee of the University of
¨
Gottingen and is conformed to the principles
embodied in the Declaration of Helsinki.
The group examined in this study consisted of
women who had recently (8–10 months) given
birth to a child in the total area of the German
capital Berlin. The coverage of neighbourhoods in
Berlin were inclusive of different economic strata
and of varying ethnic backgrounds. Each of the
12 000 families involved was informed once about
the free drinking water analysis and the study by
means of a leaflet. To guarantee prompt answer
and analysis of the water samples, the leaflets were
sent to the families, in several steps each time,
informing usually 2000 families simultaneously.
The addresses were kindly provided by the federal
resident’s registration office of Berlin (selected by
birthday). After a written declaration of consent
had been obtained and the infants were approxi-
 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
mately 8 to 10 months of age, all families willing
to participate were asked to collect two different
composite samples of drinking water in rinsed
150-ml PE bottles sent to them by post. Composite
sample type 1 was collected by taking an aliquot
of 100 ml of tap water each time it was used in
the household. On the next day, collection of 250
ml of tap water in the morning, at noon, in the
evening, and before going to bed was required for
composite sample type 2 (general course of the
day). The collected water for both sample types
had to be stored during the day in a clean non-
metal container of the household until the PE
transport bottles were filled with the composite
samples in the evening. The families were instruct-
ed to cover the container between each collection.
In the initial phase of the study, only one screening
sample taken by the collection of a small aliquot
of tap water filled directly into the 150-ml PE
bottles each time tap water was used for baby food
preparation was tested. Additional composite sam-
ples type 1 and 2 were collected if 0.5 mg copper
per liter were reached or exceeded in the house-
hold’s screening samples and the infant was not
fully breastfed.
For all households, a questionnaire served to
asses factors of interest regarding installation,
building and uptake of tap water. Tap water sam-
ples were collected between June 1998 and March
2001. Due to practical reasons and remaining
uncertainties in food copper concentrations, a
detailed feeding history was not recorded. A socio-
economic analysis of the families has not been
made.
Following collection in rinsed 150-ml PE bot-
tles, samples were acidified at pH-0.2 with nitric
acid. The copper concentration of the water sam-
ples was measured by atomic absorption spec-
trometry using a graphite furnace Perkin–Elmer
SIMAA 6000 following German industrial stan-
dard DIN 38 406-7 (wavelength: 324.8 nm, detec-
tion limit: 0.005 mg Cuyl). For internal quality
control, the reference material Perkin–Elmer Qual-
ity Control Standard-21 elements PE pure was
used. The laboratory has successfully participated
in regular external quality assurance for all meas-
ured parameters.
129
For some households pH value, conductivity,
total hardness, SO2y 1y 3y
4 -, NO3 -, PO4 - and F
1y
concentrations were determined from an additional
flushed water sample (water was allowed to run
from the tap until there was a noticeable difference
in its temperature, then 500 ml was collected). Ion
chromatography was used to analyse the water for
SO2y 1y 3y
4 -, NO3 -, PO4 - and F-concentrations. A
detailed description of used analysis methods can
be found in Zietz et al. (2001).
A family with copper concentrations of 0.8 mgy
l or higher in one of the composite samples was
recommended to have a paediatric examination if
their infant had received 200 ml of tap water per
day or more for at least 6 weeks. The copper limit
of 0.8 mgyl has been chosen for this study because
this value nearly equals the average copper content
in human milk (0.72 mgykg; Souci et al., 1994).
If the copper concentration exceeded 2.0 mgyl
(limit of the WHO), a paediatric examination was
recommended even for exposures below 200 ml
per day. All other families with elevated copper
concentrations in their composite samples were not
recommended to have a paediatric examination of
their infant. Each of the paediatricians involved
was notified about the background and aim of the
study. The paediatric examination was recommend-
ed to include the drawing of a blood sample and
an examination of the liver using palpation and
ultrasound imaging. During the blood serum anal-
ysis the following parameters were determined:
serum copper (colorimetic assay with PAESA,
Nobis, Endingen, Germany); ceruloplasmin (neph-
elometry with rabbit antiserum, Behring, Marburg,
Germany); immunoglobulins (IgG, IgM, IgA; tur-
bidimetry with rabbit antisera, Behring); glutamate
oxaloacetate transaminase (GOT); glutamic pyru-
vic transaminase (GPT); gamma-glutamyl tran-
speptidase (GGT) (enzymes measured with
photometric assay, Beckman Coulter, Galway, Ire-
land); total bilirubin (colorimetric assay with diazo
reagent, Beckman Coulter); and CRP (turbidime-
try; Beckman Coulter). Immunoglobulins and CRP
were measured to detect signs of infections that
may alter the other serum parameters. For ethical
and practical reasons a control group for serum
values was not suitable.
130 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144

Statistical analysis of the collected data was Table 1

performed with the software MS Excel䉸 2000, Statistic overview of composite samples type 1 and 2 taken
independently of screening samples
WinStat䉸 (3.1 and 2000.1) and SPSS 10.0.7䉸.
Values of copper concentrations in tap water below Composite Composite
the confidence limit (0.01 mgyl) were included in sample sample
the statistics as 0.009 mgyl. type 1 type 2
Number of samples 2619 2619
3. Results Mean 0.436 mgyl 0.561 mgyl
Maximum 3.5 mgyl 4.2 mgyl
3.1. Copper concentrations and physicalychemical Standard deviation 0.419 0.487
5th percentile 0.02 mgyl 0.02 mgyl
parameters 10th percentile 0.04 mgyl 0.05 mgyl
25th percentile 0.12 mgyl 0.19 mgyl
In total, water samples of 2944 households were Median 0.32 mgyl 0.45 mgyl
sent back by the families and analysed for copper 75th percentile 0.65 mgyl 0.80 mgyl
concentrations in our laboratory. A written decla- 90th percentile 0.96 mgyl 1.20 mgyl
95th percentile 1.25 mgyl 1.42 mgyl
ration of consent was in total returned by 4404 99th percentile 1.90 mgyl 2.18 mgyl
households, but of these 1460 households (33.2%)
could not be recruited for tap water sampling
because they withdrew from the study or moved correlation between copper concentrations in com-
without leaving a forwarding address. In the initial posite sample type 1 and 2 was 0.85.
phase of the study, screening samples were col- Results of chemical and physical parameters
lected in 325 households (115 families withdrew tested in selected households located in different
form the study in this phase). In 145 screening suburbs of Berlin can be found in Table 2. Meas-
samples, a copper concentration of 0.5 mgyl or ured PO3y 4 -concentrations in these households
higher was found. All affected families having not were in all cases below 0.1 mgyl. Maximum
fully breastfed infants (102 families) were asked NO1y3 -concentration was 3 mgyl.
to additionally collect composite samples type 1 In the complete study, 468 (17.4%) composite
and 2. Of these, 68 families sent back composite samples type 1 and 738 (27.5%) composite sam-
samples and 37 had copper concentration of 0.8 ples type 2 had measured copper concentrations of
mgyl or higher in one or both of these composite 0.8 mg or higher including composite samples
samples. The mean copper concentration of all collected after elevated copper concentrations in
screening samples was 0.58 mgyl and the median screening samples had been found. In total, 804
0.42 mgyl. households (29.9%) had concentrations of this
In the 2619 collected households’ composite heavy metal in one or both of the sample types
samples taken independently of screening samples, equal or higher than 0.8 mgyl.
the mean copper concentration was 0.44 mgyl in Regarding the material of the domestic installa-
the composite samples type 1 and 0.56 mgyl in tion, 1509 families stated to have copper pipes
type 2. Other statistical results of measured copper and 861 explicitly stated to have no copper pipes
concentrations of these composite samples can be (63.7% of all definite statements said to have a
found in Table 1. The highest values in the copper installation). The remaining households did
collected composite samples were 3.5 mgyl for a not know or did not make a statement about this
type 1 sample and 4.2 mgyl for a type 2 sample. point at all. There was a relationship between
It can be seen that copper concentrations in the mean and median copper concentrations and age
general course of the day (composite sample type of the installation (construction or renovation of
2) tend to be higher. The copper concentrations in the pipes). Measured concentrations fell with
composite sample type 1 and 2 were not normally increasing age of the water pipes until the age
distributed and could not be transferred to nearly class of 35–40 years and then rose again (Fig. 1).
match such a distribution. The Spearman rank Assessing only households that explicitly stated to
 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144 131

Table 2
Chemical and physical parameters of selected households located in different suburbs of Berlina

ID-No. Suburb pH Conductivity Total pHc- SO2y

4 Cl1y F1y
value (mSycm) hardness value (mgyl) (mgyl) (mgyl)
(8dH)
3341 Charlottenburg 7.68 862 19.2 7.5 145 75 0.18
3342 Hellersdorf 7.41 768 17.6 7.5 139 56 0.19
3344 ¨
Kopenick 7.45 770 17.2 7.6 137 60 0.19
3346 Prenzlauer Berg 7.43 644 15.5 7.6 77 33 0.20
3498 Prenzlauer Berg 6.8 665 15.4 7.1 87 35 0.18
3343 ¨
Schoneberg 7.65 793 18.2 7.5 117 62 0.17
3340 Spandau 7.40 667 15.1 7.5 73 44 0.12
3345 Weißensee 7.63 794 19.4 7.5 148 74 0.19
Mean 7.4 745 17.2 7.5 115 55 0.18
a
Households were selected based on their location in Berlin not due to their copper concentrations. ID-No. 3340, 3343, 3344
and 3346 had copper concentrations elevated above 0.8 mgyl in one or both of their composite samples.

have a copper installation, this relationship was
also clearly visible and measured concentrations
fell until the age class of 40–45 years. There was
a slightly irregular decline and increase of mean
concentrations compared to values of all house-
holds (data not shown).
Gender relation of the last born infant stated by
participating families was very close to 1 to 1
(excluding twins and triple births). This indicates
that at least for this parameter there was no
significant difference in the motivation of families
to participate in the study.
3.2. Health status of exposed infants
In total, a paediatric examination was recom-
mended for 541 infants. Nine families fulfilling
the requirements for a recommendation of a pae-

Fig. 1. Mean copper concentrations and stated age of installation in composite sample type 1 and 2 (excluding samples collected
in relation to screening samples).
132 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
Fig. 2. Frequencies of all measured infant values of serum GOT, GPT and GGT grouped in classes.
diatric examination could not be contacted any-
more. Nearly every of these exposed infants were
inspected and examined by a physician; in many
cases during regular examinations recommended
for every infant and child in Germany. Only in six
cases was a medical examination completely
refused. In one case, an examination was not done
because the paediatrician had found that there was
actually no exposition of the infant. In eight other
cases, a result of a possible medical examination
could not be acquired.
None of the examined infants showed any symp-
tom of liver damage such as icterus or frequent
vomiting. There was no clinical suspect of a
paediatrician that a child is suffering form a liver
disease. Results of inspection and liver palpation
were only evaluated by the local paediatricians
(no external standardisation). In total, 183 infants
and children received a paediatric examination
including a blood serum analysis (Fig. 2). Of these
blood samples, 124 were analysed in the laboratory
¨
of the University of Gottingen. In one of these
examined infants, the copper value in a composite
sample of the household exceeded 3 mgyl and in
another case equalled 3.0 mgyl). Of 183 infants
examined with a blood serum analysis, eight had
never been breastfed and in total 34 had received
drinking water beginning with their birth.
One blood serum analysis of an infant (ID No.
B4804) was excluded from statistics and depiction
in the tables because by mistake a full-blood
sample was sent to our laboratory showing severe
haemolysis. A second blood sample was refused
by the mother. Physical examination and ultra-
sound imaging of the liver gave no indication of
a liver disease. The child had received an estimated
800 ml of tap water daily beginning at the 35th
week (copper concentrations in tap water: com-
posite sample type 1: 0.85 mgyl; and type 2: 1.05
mgyl). Additionally a second case was also
excluded from statistics and depiction in the tables.
This infant (ID No. B4759) was known to have a
congenital infection with Toxoplasma gondii
including an affection of the liver and was treated
with chemotherapeutics. In the frequent controls
of this infant, transaminases and cholinesterase
were slightly or moderately elevated in many
cases. This infant also had an increased liver
volume. The copper concentrations in this house-
hold were 1.15 mgyl in composite sample type 1
and 1.6 mgyl in type 2. The infant had received
1000 ml of this tap water per day beginning at its
19th week of life.
There were eight infants with outlying values of
GOT, GPT, GGT, or serum copper and no outlier
of total bilirubin. Values were defined as outliers
 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
if the mean of this serum parameter was exceed
by four standard deviations or if it had a distance
of the mean with a chance of occurrence below
Ps0.05. Details of these infants can be found in
Table 3. Six of these infants had clinically diag-
nosed infections at the time of the serum analysis
and one was known to have a liver haemangioma
and a ureteric obstruction including hydronephros-
is. Only in one case, there was no hint of a disease
that may be associated with the outlying serum
parameter. Although GOT, GPT, GGT are indica-
tors of liver or muscle diseases, it seems likely
that infections attribute to these minor elevations
of the parameters. The liver and the muscles can
be affected during infection and using medication
during infection can also be a cause of elevations
(Pratt and Kaplan, 2000). Serum copper is typi-
cally be elevated by infective diseases (Beshgetoor
and Hambidge, 1998). None of these outliers were
excessively elevated above the used reference val-
ues. Three of these infants had received a serum
control analysis (B990, B2437a, B2437b). In all
cases, these infants had an infection at the time of
the first blood sample. Two of these (B2437a,
B2437b) had much lower values in the serum
controls. One infant (B990) did not have normal
values of GOT, but it was infected again at the
time of the control sample. Details of other infants
that received a serum control were given in Table
3, too.
In five cases (slightly) unusual ultrasound imag-
es of the liver or spleen were found (serum
parameters and a detailed exposition history are
also given in Table 3). Only elevations of serum
copper (no definitive reference value available;
Kirsten et al., 1985), total bilirubin, GOT, GPT,
GGT and ceruloplasmin were mentioned in the
following (Witt and Trendelenburg, 1982; Thomas,
1984). None of the examined infants showed any
symptom of liver damage such as nausea or vom-
iting. An 11-month-old girl had a slight hepato-
megaly (ID B802) with a liver length of 9.5 cm
that is 0.5 cm above the reference value of this
body length class. Serum copper, total bilirubin,
GOT, GPT, GGT and ceruloplasmin were in the
reference ranges. In another case, an 11-month-old
girl (ID B799) showed a slightly enhanced echo-
genicity throughout the liver with ultrasound imag-
133
ing. Additionally, bile ducts in the liver were
slightly rarefied. Serum values of copper, GOT,
GPT, GGT were in the reference ranges and the
total bilirubin value (1.0 mgydl) was at the upper
reference limit (Witt and Trendelenburg, 1982).
Three other infants were diagnosed to have a slight
splenomegaly. Two of these three cases of slight
splenomegaly are clearly associated with an infec-
tive disease. It is very probable that the infection
was the reason for the (transient) splenomegaly in
two of these cases. Infections are a known cause
of elevated spleen size (O’Reilly, 1998), due to its
immunologic function. These are, therefore, the
most likely cause of these slightly abnormal results
in these cases.
3.3. Statistics using results of serum analysis
Based on the Kolmogorov–Smirnov test only
serum copper and IgG were normally distributed
among all measured serum values of exposed
infants. Different daily and total copper intakes
through tap water were calculated for an examined
infant by separately using copper concentrations
of composite sample type 1 and 2 and a mean of
both types combined with parent’s statements of
volume and duration of tap water ingestion. The
different estimated daily and total copper intakes
were not normally distributed. Therefore, the
Spearman rank correlation between estimated
intakes and serum parameters was calculated (one-
sided and two-sided significance). No significant
correlation of GOT, GPT, GGT, total bilirubin,
serum copper, or ceruloplasmin and estimated
copper intake through tap water could be found
except one. Assuming a one-sided significance
(P-0.05; prerequisite: direction of the effect is
known) in one case there was a significant rank
correlation of y0.133 between serum copper and
daily copper intake calculated with composite sam-
ple type 1. This correlation could not be found
with all other estimated daily and total copper
intakes or using a two-sided significance Fig. 3.
In a next step, complete records of infants
including outliers in one serum parameter that
exceed the mean by four standard deviations or
that had a distance of the mean with a chance of
occurrence below Ps0.05 were eliminated. No
 134
Table 3
Results of blood serum analyses of infants exposed to elevated copper concentrations having outliers in their serum parameters (serum copper, GOT, GPT, GGT) or
unusual ultrasound images of the liver or spleen or serum analyses that were repeated

B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144 135

Table 3 (Continued)
 136
Table 3 (Continued)

B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
Used reference values: GOT 2nd–12th month -28 mUyml, )12 months -23 mUyml (Witt and Trendelenburg, 1982); GPT 2nd–12th month -31 mUyml, )
12 months -25 mUyml (Witt and Trendelenburg, 1982); GGT 2nd–12th month up to 100 mUyml, )12 months -21 mUyml (Witt and Trendelenburg, 1982); total
bilirubin 2 months and older: up to 1 mgydl (Witt and Trendelenburg, 1982); Ceruloplasmin 15–60 mgydl (Thomas, 1984); IgM 7th–12th month 0.36–1.04 gyl, 2
years 0.72–1.60 gyl (Uffelmann et al., 1970); IgG 7th–12th month 3.5–11.8 gyl, 2 years 5.2–10.8 gyl (Uffelmann et al., 1970); IgA 7th–12th month and 2 years
0.36–1.65 gyl (Uffelmann et al., 1970); CRP up to 1 mgydl (Sabel and Wadsworth, 1979). Serum copper: 6th–12th month mean 136–161 mgydl, standard deviation
24–32 mgydl (Kirsten et al., 1985). n.t.sNot tested; B2437a and B2437b are twins.
a
Blood serum tested in foreign laboratory.
 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144 137

Fig. 3. (a) Serum GOT and estimated total copper intake through tap water (calculated with copper concentrations from composite
sample type 1) including linear trend line (Excel䉸 ). Spearman rank correlation y0.090 with Ps0,228 (2-tailed). (b) Serum GOT
and estimated daily copper intake through tap water (calculated with copper concentrations from composite sample type 1) including
linear trend line. Spearman rank correlation y0.060 with Ps0.422 (2-tailed). (c) Serum copper and estimated total copper intake
through tap water (calculated with copper concentrations from composite sample type 1) including linear trend line. Spearman rank
correlation y0.071 with Ps0.357 (2-tailed). (d) Total bilirubin and estimated total copper intake through tap water (calculated
with copper concentrations from composite sample type 1) including linear trend line. Spearman rank correlation 0.101 with Ps
0.201 (2-tailed).

significant correlation of GOT, GPT, GGT, total
bilirubin, serum copper, or ceruloplasmin and esti-
mated copper intake (daily and total intakes)
through tap water could be found after eliminating
these records. The same statement is valid if only
outliers of GOT, GPT, GGT, serum copper, or
ceruloplasmin were eliminated (there were no
outliers of total bilirubin).
Additionally data of male (ns102) and female
(ns81) infants were evaluated separately. Also
subgroups including only infants whose blood
samples were taken not later than 14 days (ns
53) or in another group 21 days (ns81) after
notification about the elevated copper tap water
concentrations were evaluated separately, too. This
was done because some parents may have stopped
feeding their infants with this tap water and a
possible health effect may only be effective for a
limited time. In these groups, several significant
correlations could be seen (Spearman rank; P-
0.05; one-sided significance) of GOT, GPT, GGT,
serum copper, or total bilirubin and different daily
and total copper intakes through tap water. In all
cases, these correlations were weak ()0.30) and
in most cases they had a negative sign. This would
mean that higher copper intake would lead to
lower serum parameters which is the opposite of
the expected effect. In the subgroup of male
138 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144

infants, total bilirubin was found to significantly Table 4

(Spearman rank; P-0.05) and positively correlate Means of serum parameters in three different exposition groups
(total copper intake with tap water) calculated for all serum
with the daily and total copper intakes estimated parameters (ns183), serum parameters measured in the Got- ¨
with the means of composite samples type 1 and tingen laboratory (ns124) and all serum parameters excluding
2 in two cases. These correlations were both very outliers (ns175)
weak ()0.20).
For further evaluation of serum values, all tested All measured serum parameters
infants were divided in three groups according to Lowest Medium Highest
their estimated daily and also total copper intakes exposition exposition exposition
through tap water (in every case calculated with group group group
copper concentrations in sample type 1). For this Serum copper (mgydl) 135.17 129.52 126.76
reason quantile function (0.33 and 0.66) of MS GOT (Uyl) 27.07 22.27 22.69
Excel䉸 was used. This was also done for records GPT (Uyl) 15.24 12.82 13.48
GGT (Uyl) 7.17 6.15 6.23
excluding laboratories other than the laboratory of Total bilirubin (mgydl) 0.41 0.45 0.50
¨
the University of Gottingen and as a third one IgM (gyl) 0.89 0.73 0.75
records excluding outliers as defined above. Fol- IgG (gyl) 4.58 4.82 4.34
lowing that, values of the highest and lowest group Ceruloplasmin (mgydl) 29.56 32.98 28.50
were compared using the U-test (Mann–Whitney). Free copper (mmolyl) 7.24 5.37 6.97
There was no case for the parameters, serum ¨
Serum parameters only measured in the Gottingen laboratory
copper, GOT, GPT, GGT, total bilirubin, IgM or Serum copper (mgydl) 139.18 125.57 130.58
GOT (Uyl) 24.27 22.82 23.02
ceruloplasmin where values of the highest expo- GPT (Uyl) 15.15 13.36 14.55
sure group were significantly higher (P-0.05) GGT (Uyl) 6.54 6.00 6.38
than that in the lowest exposure group. This trend Total bilirubin (mgydl) 0.47 0.50 0.56
can also been seen in Table 4, depicting mean IgM (gyl) 0.82 0.70 0.79
values in the three exposure groups. IgG (gyl) 4.35 4.24 4.43
Ceruloplasmin (mgydl) 27.48 28.16 27.85
Because the hypothesis, that infants with high Free copper (mmolyl) 9.00 7.14 7.87
copper intake through tap water develop mild signs
of liver abnormality, has not been investigated so All serum parameters excluding outliers
Serum copper (mgydl) 130.70 128.48 128.04
far there is data on incidence, etc. Therefore the GOT (Uyl) 23.98 22.53 22.50
power of this test can only be calculated with GPT (Uyl) 13.29 12.96 13.46
assumptions. For example, a sample size of 61 GGT (Uyl) 6.24 6.23 6.23
exposed and 61 unexposed persons (equalling Total bilirubin (mgydl) 0.42 0.46 0.49
highest and lowest exposure group size) and a IgM (gyl) 0.85 0.73 0.76
IgG (gyl) 4.53 4.84 4.40
confidence level of 95% would result in a test Ceruloplasmin (mgydl) 29.75 32.87 28.79
power (1-b) of 80% assuming a frequency of
2.5% in the unexposed group and 20% in the Remarks: total copper intake estimated with copper concen-
trations in composite samples type 1; in the third part of the
exposed group (calculation with EpiInfo 2000). table records of infants with outliers of serum copper, GOT,
In a further step, the serum concentration of GPT, GGT, ceruloplasmin were excluded completely (there
ceruloplasmin-bound copper (‘free’ copper) was were no outliers of total bilirubin); calculation method of free
estimated to be the difference of total serum copper copper is described in the text.
(mmolyl) subtracted by six times the amount of
ceruloplasmin (mmolyl) (Eife et al., 1999). This It should be kept in mind that this calculation
calculation is based on the assumption that one could include the problem that nephelometry, being
molecule ceruloplasmin binds six atoms of copper an immunologically-based method, may overesti-
and, second, the molecular weight of ceruloplas- mate the concentration of ceruloplasmin. There-
min is 132 000 daltons. This calculated free copper fore, the non-ceruloplasmin-bound Cu would be
value can either be expressed as absolute value in underestimated. Altogether, 133 values of free
mmolyl or as the percentage of total serum copper. copper could be calculated from our data with a
 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
mean absolute value of 6.53 mmolyl (median 6.62
mmolyl). Expressed as percentage of total serum
copper, there was a mean of 30.6% (median
33.7%). The 90th percentile was 11.49 mmolyl for
the absolute value and 48.4% expressed as relative
value. Spearman rank correlation between different
estimated daily and total intakes (as described
above) and free copper values were calculated. No
significant correlation could be found. Beside a
correlation of serum copper or ceruloplasmin and
free copper values there were weak rank correla-
tions ()0.22) between relative free copper values
and GPT or total bilirubin (P)0.025). Using the
U-test (Mann–Whitney) to compare the highest
and lowest of three exposure groups under differ-
ing circumstances as described above gave no
significant differences for free copper values.
4. Discussion
Copper concentrations found in this study are
relatively high compared to that of other regions.
A similar study of this institute in the area of
¨
Gottingen, Germany, showed lower copper concen-
trations in tap water (Dassel de Vergara et al.,
1999 and unpublished results). The mean copper
¨
concentration in the Gottingen study was 0.18 mgy
l in the 1619 collected stagnation samples and
0.11 mgyl in the 1660 random daytime samples.
In Western Germany in the years 1985–1986, a
mean copper concentration of 0.248 mgyl (median
0.067 mgyl) was measured in stagnated water
samples collected in sampling places throughout
the country in 2577 households (Krause et al.,
1991). In random daytime samples, a mean of
0.154 mgyl and a median of 0.04 mgyl was found.
They could observe the trend that rural areas had
lower copper values than urban areas ()100 000
inhabitants). In water samples drawn in water
supply facilities (before entering the distribution
network, ns97) a mean of 0.013 mg copper per
litre and a median of 0.005 mgyl was measured.
Similar results were found in the years 1990–1992
for the total area of Germany (Becker et al., 1997).
In Uppsala and Malmo, ¨ Sweden, concentrations
of unflushed water samples (equivalent in most
cases to stagnated water samples) were analysed
for 1178 children by Pettersson and Rasmussen
139
(1999). The median copper concentration was 0.72
mgyl and the 90th percentile 2.11 mgyl. The
median concentration was 0.81 mgyl in Malmö
and 0.54 mgyl in Uppsala. Among the 430 children
who participated in this part, the mean daily intake
of water was 620"200 ml. An in-depth diary
study investigated the effect of flushing on copper
values. In this part, the median copper concentra-
tions were 0.93 mgyl in unflushed drinking water,
0.19 mgyl after flushing for 30 s and 0.08 mgyl
after flushing until the water ran consistently cold.
Falling median copper concentrations with increas-
ing age of the installation (due to generation of a
surface layer slowing down corrosion) could be
proved in this study, too. Measured concentrations
in our study of the Berlin area fell with increasing
age of the water pipes until the age class of 35–
40 years and then increased again. This trend was
also clearly visible assessing only households that
explicitly stated to have a copper installation. The
rising concentrations in higher age classes may be
caused by an increasing probability of repair and
partial replacement (or unknown total replace-
ments) of the installation in higher age classes.
The comparison of the cited investigations points
out that (mean) copper concentrations in the user’s
tap water can vary in a broad range. The Berlin
water supply facilities do not add anticorrosive
substances (for example based on a silicate or
phosphate) to their production of water (personal
communication, Berliner Wasserbetriebe, 2001).
In documented cases of NICC (Dieter et al.,
1999; Eife et al., 1991; Schafer¨ ¨
and Schumann,
¨
1991; von Muhlendahl and Lange, 1994; Schim-
melpfennig et al., 1997; Walker-Smith and Blom-
field, 1973) in which the liver disease was
supposedly caused through tap water, but where
data could be gathered only retrospectively, the
copper concentrations of the tap water were found
to lie between 1 and 3 mgyl and were occasionally
as high as 26.4 mgyl. Copper seems, therefore, to
be involved in the aetiology of NICC. The con-
centration range of a few milligrams of copper per
liter water was reached in several cases in this
investigation. For example 40 households had
copper concentrations of 2 mgyl or higher in
composite sample type 2.
140 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
Aminotransferase (GOT and GPT) levels are
sensitive indicators of liver-cell injury and are
helpful in recognising hepatocellular diseases such
as hepatitis (Pratt and Kaplan, 2000). Liver func-
tion indicators such as albumin and clotting factors
(e.g. prothrombin time) are a component of grad-
ing systems for chronic liver diseases. They do
not have a good performance as a screening test
(Rochling, 2001). Both aminotransferases are nor-
mally present in serum at low levels (Pratt and
Kaplan, 2000). Because blood serum analyses of
infants examined in this study were also made in
several other laboratories in Germany, we know
that reference values of aminotransferases vary
widely among laboratories ()25%). This is also
described in the literature (Pratt and Kaplan,
2000). The normal range for any laboratory test is
the mean value in a group of healthy persons "2
standard deviations. Thus, 5% of the results
obtained from these group can be expected to be
outside the defined normal range and 2.5% above
the upper limit of normal (Pratt and Kaplan, 2000).
Data to define reference limits in infants and
children are often limited in number of available
studies and number of examined persons. For
example, used reference values of GOT, GPT and
GGT are based on analyses of 210–254 infants
and children measured in 15 different paediatric
hospitals (Witt and Trendelenburg, 1982). It can
be seen that our study has tested nearly as much
infants and children (ns183). This fact is also
true for C-reactive protein (CRP), immunoglobu-
lins (Sabel and Wadsworth, 1979; Uffelmann et
al., 1970) and serum copper (no definitive refer-
ence values; Kirsten et al., 1985). This is in many
cases aggravated by the fact that measured values
in infants and children vary substantially with the
age (Kirsten et al., 1985). Because of these reasons
we do not discuss every excess of a reference limit
in detail. Nearly all of these elevations are mar-
ginal.
We, therefore, conclude that the higher than
expected number of infants with slightly elevated
aminotransferase levels referring to cited literature
does probably not reflect a pathologic status of the
tested infants. This assessment is confirmed by the
fact that no positive correlation between amino-
transferase levels and the amount of copper ingest-
ed with drinking water or the copper concen-
trations in water was found.
It is recommended as a first step in the evalua-
tion of a patient with elevated liver enzyme levels,
but no symptoms, to repeat the test to confirm the
result. If the result is still abnormal, the physician
should evaluate the degree of the elevation. A
minor elevation (less than twice the normal value)
may be of no clinical importance if common
disorders have been ruled out and, in fact, may
not even be abnormal (Pratt and Kaplan, 2000).
While working on the study, we used reference
values based on several studies (Witt and Trende-
lenburg, 1982; Sabel and Wadsworth, 1979; Uff-
elmann et al., 1970; Thomas, 1984). A repeat of
the serum analysis was recommended to the phy-
sicians if two parameters possibly relevant to liver
diseases (GOT, GPT, GGT, total bilirubin, serum
copper, ceruloplasmin) were outside the reference
range or one parameter was elevated more than
20% above the reference limit. In many cases, the
paediatricians having more information about these
infants including the results of their clinical exam-
ination did not confirm this recommendation. Gen-
erally, the following hepatic causes of chronically
elevated aminotransferase levels (GOT, GPT) are
known in adults: alcohol abuse; medication; chron-
ic hepatitis B and C; steatosis and non-alcoholic
steatohepatitis; autoimmune hepatitis; hemochro-
matosis; Wilson’s disease (in patients F40 years
old); and alpha1-antitrypsin deficiency (Pratt and
Kaplan, 2000). Non-hepatic causes are celiac
sprue, inherited disorders of muscle metabolism,
acquired muscle diseases and strenuous exercise
(Pratt and Kaplan, 2000). Some of these diseases
can be excluded in infants and other possible
diagnoses such as NICC have to be added.
Slightly unusual ultrasound images were found
in two cases of the liver (slight hepatomegaly;
slightly enhanced echogenicity and bile ducts
slightly rarefied) and in three cases of the spleen.
Ultrasound of the liver is used as a screening
imaging tool in cases of patients suspicious for
diffuse liver disease and is helpful in the term of
follow-up examinations (Hammerstingl et al.,
2001). In the case of liver cirrhosis, there are
 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
variable signs of homogenicity and echogenicity
changes. Hepatomegaly and enhanced echogenici-
ty can be signs of a pre-stage or an early stage of
liver cirrhosis. On the other hand, patterns of
enhanced echogenicity are unspecific and can be
caused by different disorders of toxic agents (Ham-
merstingl et al., 2001). In case of NICC, the
pathologic pattern of a micro-nodular cirrhosis is
to be expected, but in early stages a more subtle
and rather unspecific liver histopathology may be
present (Muller
¨ et al., 1998). In all cases of NICC
for which data are available, biochemical markers
of hepatic injury such as aminotransferases, alka-
line phosphatase, bilirubin, albumin and prothrom-
bin time have been shown to be abnormal (Muller ¨ intake of the infants trend to be lower than in the
et al., 1998). In both described cases of abnormal
liver images in this study, serum analysis did not
reveal any relevant excess of reference values and
no clinical symptoms were seen. Three other
infants were diagnosed to have a slight spleno-
megaly. In two of these three cases, it is very
probable that clinically diagnosed infections were
the reasons for the slight splenomegaly. Spleno-
megaly is known to be a concomitant symptom of
liver cirrhosis, but as a detached symptom it does
not give proof of liver disease, because several
other systemic diseases such as haematological,
infectious, congestive, or inflammatory disorders
are known reasons, too (O’Reilly, 1998). Addi-
tionally, reference values in ultrasonic imaging of
infants and children are only based on small
examination groups similar to the situation in
laboratory medicine, and results are influenced to
some extent by personal experience and individual
interpretations of images (Konus et al., 1998;
Rosenberg et al., 1991; Bowie, 2000). Therefore,
we conclude that these slightly abnormal results
in ultrasound imaging are, with a high probability,
not a sign of liver disorder.
We could find no supporting facts concerning
the hypothesis that an existing health effect may
have disappeared because many parents could have
stopped feeding their infants with copper contam-
inated tap water and that, secondly, a possible
health effect may only be effective for a limited
time. Assessing only infants statistically whose
serum samples were examined no later than 14
141
days, or in a second group no later than 21 days,
no positive correlation of daily and total copper
intakes and serum parameters could be found. In
all cases, an infant was examined including a
serum analysis, and we tried to contact the families
again to get information whether tap water was
used for the infant after the notification about the
elevated copper concentrations. Most of the
responding parents stated that they further used
the tap water for their infants but many of them
flushed the water before using it for their infants.
It is known that flushing water leads to lower
coppers concentrations (Pettersson and Rasmus-
sen, 1999). So it can be assumed that copper
weeks before notifying parents, but the detailed
amount of this copper intake remains unknown.
So there remains an uncertainty in estimating the
copper intakes but using data of subgroups gives
no hint that this would effect the results of the
study.
We can summarise from this study that from the
paediatric examination (physical examination and
blood test) of the infants whose food had been
prepared using tap water with a copper concentra-
tion of 0.8 mgyl or more (measured maximum 4.2
mgyl), no confirmed indication of a liver malfunc-
tion could be found. None of the examined infants
showed symptoms of liver damage. Additionally
no negative health effects could be found in the
statistical analysis of the serum parameters serum
copper, GOT, GPT, GGT, total bilirubin, or ceru-
loplasmin in relation to different estimated daily
and total copper intakes of the infants from tap
water.
In an experimental study by Olivares et al.
(1998), no signs of disease were found in a group
of infants whose baby food had been prepared
with water having copper concentrations of 2 mgy
l. But it has to be taken into account that the
infants in the study of Olivares et al. (1998) were
breastfed until they reached 3 months of age. Only
at that time, exposition of the infant with copper
supplemented drinking water started. This is in
contrast to many observed cases of NICC, that
were breastfed for a short time only or not breast-
fed at all (Dieter et al., 1999). In our study of 183
142 B.P. Zietz et al. / The Science of the Total Environment 302 (2003) 127–144
infants examined with a blood serum analysis, 34
had received drinking water beginning at their
birth. Another support of these study results is
implicated in a survey of three cities in Massachu-
setts. No fatality through liver disease was reported
for children under 6 years of age despite high
copper levels (between 8.5 and 8.8 mgyl) in tap
water and 64 124 child years of exposure (Schein-
berg and Sternlieb, 1994). This led Scheinberg and
Sternlieb to propose that a rare genetic defect may
be at the root of this form of early childhood
cirrhosis. Up until now this hypothesis remains to
be confirmed (Muller¨ et al., 1999b). In summary,
based on our present knowledge, probably a com-
bination of endogenic and exogenic factors rather
than a monocausal aetiology is responsible for the
development of ICC or NICC.
For public health purposes, a new limit for
copper of 2.0 mgyl (in composite samples) was
introduced on a European basis in 1998 (European
Union. Guideline 98y83yEG). This limit is equiv-
alent to the WHO recommendations of 1993
(World Health Organization, 1993, compare with
Fitzgerald, 1998). This guideline has recently been
transferred into German law (TrinkwV, 2001) and
most parts of this guideline including the new
copper limit will get valid at the beginning of the
year 2003. The German Drinking Water Regulation
of 1990 (TrinkwV, 1991) had specified 3 mgyl as
limit for stagnant water in houses older than 2
years. With this new approach, Germany will get
a threshold value instead of the old guideline for
copper in tap water. This threshold value will be
valid in composite samples that are more represen-
tative to consumption than stagnated water sam-
ples. The results of this study support the
assumption that the copper threshold value of 2
mgyl is preventing infants from a health hazard.
As a measure to get lower concentrations of heavy
metals, water supply facilities have the option to
add anticorrosive substances (based on a silicatey
phosphate) to their production of water. Since the
copper values of the stagnation samples were
usually somewhat higher, it follows that an infant’s
copper intake may be reduced by not using stag-
nant water in the preparation of baby food, in
particular when the pipes are new.
Acknowledgments
The authors wish to thank the German Federal
Environmental Agency (Umweltbundesamt, Ber-
lin; UFOPLAN No. 296 61 116) for providing
most of the funding for this study and all partici-
pating paediatricians for kindly supporting this
study. The authors also wish to thank C. Behmke,
S. Irmer, Dr A. Heutelbeck, Dr G. Gahnz for their
support, U. Richter for the analysis of the blood
samples and Prof. Dr Stefan Wellek (Mannheim)
for reviewing the statistical evaluation. Finally, we
want to thank all participating physicians in Berlin
for their kind support of the study.
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