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Photos
of some birth defects reported from Fallujah General Hospital between
May and August 2010. a Gastroschisis, b Hydrocephalus, c Encephalocele, d
Macrocephaly, e Spina Bifida; f Cleft lip and palate
Abstract
Between
October 1994 and October 1995, the number of birth defects per 1,000
live births in Al Basrah Maternity Hospital was 1.37. In 2003, the
number of birth defects in Al Basrah Maternity Hospital was 23 per 1,000
live births. Within less than a decade, the occurrence of congenital
birth defects increased by an astonishing 17-fold in the same hospital. A
yearly account of the occurrence and types of birth defects, between
2003 and 2011, in Al Basrah Maternity Hospital, was reported. Metal
levels in hair, toenail, and tooth samples of residents of Al Basrah
were also provided. The enamel portion of the deciduous tooth from a
child with birth defects from Al Basrah (4.19 μg/g) had nearly three
times higher lead than the whole teeth of children living in unimpacted
areas. Lead was 1.4 times higher in the tooth enamel of parents of
children with birth defects (2,497 ± 1,400 μg/g, mean ± SD) compared to
parents of normal children (1,826 ± 1,819 μg/g). Our data suggested that
birth defects in the Iraqi cities of Al Basrah (in the south of Iraq)
and Fallujah (in central Iraq) are mainly folate-dependent. This
knowledge offers possible treatment options and remediation plans for
at-risk Iraqi populations.
Keywords: Iraq, Metal exposure, Human birth defects, Folate-dependent birth defects
It
is old knowledge that exposure to chemicals can harm human
reproduction. Ancient Romans were aware that lead (Pb) poisoning can
cause miscarriage and infertility (Gilfillan 1965; Retief and Cilliers 2006).
Today it is well established that human pregnancy and fetal development
are susceptible to parents’ environmental exposure to chemical,
biological and physical agents (Mattison 2010).
A new concept of the developmental origins of health and disease has
also emerged which is defined as the process through which the prenatal
environment, or the environment during infancy, shapes the long-term
control of tissue physiology and homeostasis (Barker 2004).
We know that even slight perturbations caused by chemical exposures
during sensitive periods of fetal development can lead to increased
risks of disease throughout the life of an individual (Sutton et al. 2010).
Pregnant
mothers and their growing fetuses are especially vulnerable to exposure
to pollutants. Air and water pollution, exposure to toxic metals, and
exposure to persistent and volatile organics have been linked to adverse
pregnancy and developmental outcomes (Landrigan et al. 2004; Bocskay et al. 2005).
Recently, an unusual number of birth defects, in many bombarded Iraqi
cities, has raised international concern and a few relevant studies have
been published (Alaani et al. 2011a; 2011b; Al-Ani et al. 2010).
Following bombardment, severe contamination of water, soil, and air can
occur. Metal contamination of the public after bombardment has been
reported (Jergovic et al. 2010). Jergovic et al. (2010)
examined the blood serum metal content of the Croatian population in
areas with "moderate fighting" versus "heavy fighting". They found
significantly higher levels of metals in populations from areas with
heavy fighting. Those areas had been targeted for repeated bombardments
by the North Atlantic Treaty Organization in 1991 and 1995. Various
metals are contained in US small arms ammunition, and are contained in
US bombs (Departments of the Army, the Navy, the Air Force, Joint
Technical Bulletin, 1998; US Department of the Army Technical Manual, 1990).
Intermittent
bombing of populated cities in Iraq has occurred since 1991. Most
significant was the bombardment of Fallujah, a city in central Iraq, and
Al Basrah, a city in southern Iraq. Fallujah was heavily bombed in
2004. Subsequently, unusual numbers of birth defects have been surfacing
in that city. Al Basrah was also a target of heavy bombing (December
1998, March and April 2003). Similar to Fallujah, after the 2003
invasion and occupation of Iraq, the medical staff in Al Basrah
Maternity Hospital has been witnessing a pattern of increase in
congenital birth defects. Based on these observations, many suspect that
pollution created by the bombardment of Iraqi cities has caused the
current birth defect crisis in that country (Al-Hadithi et al. 2012).
In
the present article, we have reported on 56 Fallujah families’ hair
metal levels and the kinds of birth defects documented in these
families. The Fallujah study was conducted in 2010. We have also
presented a year-to-year account of the types and numbers of birth
defects in Al Basrah Maternity Hospital from 2003 to 2011. Our aim was
to examine the populations of Fallujah and Al Basrah for possible metal
exposure. In our samples from Al Basrah, we were looking at three
tissues (hair, toenail, and teeth) and wanted to determine which tissue
provides a better medium for metal analysis.
Materials and Methods
Between
May and August 2010, 56 Fallujah families were recruited into an
epidemiological "case study" project. Cases (n = 46) and controls
(n = 10) had come to Fallujah General Hospital for delivery or
treatment. Using a questionnaire, information on reproductive history of
families and the parents’ siblings, residence history, health and
disease during pregnancy, drug use during pregnancy, smoking and alcohol
use, source of water for the family, and exposure to potential war
contaminants was collected. Hair samples were collected from all members
of the family (mothers, fathers and the children) and patient consent
was obtained at the same time. Participants had lived continuously in
Fallujah since 1991. Percentages of birth defects and miscarriages were
determined (Fig. 1);
grouping of the years was based on "before" and "after" the 2003
attacks. For the period of 2003 onward, we used 3-year intervals. The
remaining single-year data, from 2010, was added to the last group. For
each of the groups, the percentages of birth defects and miscarriages
were determined by totaling live births and multiplying that figure by
100, then dividing the value by the total number of birth defects or
miscarriages, respectively. The metal content of hair samples was
determined by inductively coupled plasma mass spectrometry (ICPMS). We
collected hair rather than blood samples, since it is difficult to
obtain and transport blood samples in a war zone.
Percentage
of birth defects and percentage of miscarriages among 56 Fallujah
families who had come to Fallujah General Hospital for treatment or
delivery between May and August 2010
In
Al Basrah, records of the Department of Obstetrics and Gynecology at Al
Basrah Maternity Hospital were examined for numbers and types of
diagnosed and reported birth defects during 1994 and then from 2003 to
2011 (Table 1).
Birth defects in the newborns were diagnosed by certified medical
doctors. Additionally, between September 2011 and January 2012,
twenty-eight families who had come to Al Basrah Maternity Hospital for
treatment or delivery were recruited into this study by local physicians
and patient consent was obtained. Hair and toenail samples were
collected from two groups: parents who recently had a child with a birth
defect (n = 14); and parents who had a normal child (n = 14). Only 6
samples of hair and 6 samples of toenails from parents of children with
defects were sufficient in weight to be analyzed for metal levels.
Yearly account of birth defects per 1,000 live births in Al Basrah Maternity Hospital from 2003 to 2011
Tooth
samples were simultaneously collected at the Al Basrah Dental School
from parents of normal children (n = 10) and parents of children with
cardiac and neural tube defects (n = 12). Only 5 of each group had
sufficient and suitable tooth tissue for metal analysis. Parents did not
smoke or drink. Two samples of deciduous teeth were collected from
children with birth deformity who had survived. Deciduous teeth from
normal children were provided by the School of Dentistry at Isfahan
University of Medical Sciences (n = 18, two samples were analyzed).
Patient/parental consent was obtained.
Hair samples’ treatment, digestion and their analysis for Fallujah samples followed Batista et al. 2009
without modification. XSERIES 2 ICP-MS (Thermo Fisher Scientific,
Germany) was used in the standard configuration, with ASX-510
auto-sampler (Cetac, USA). Instrument optimization was by auto-tune
function, when required. The instrument parameters were: RF Power (W)
1400, Cool Gas Flow (L/min) 13, Auxiliary Gas Flow (L/min) 0.8,
Nebuliser Gas Flow (L/min) 0.85–0.90, Sample Uptake Rate (mL/min) 0.4
approx., Sample Introduction System Concentric nebuliser with low-volume
impact bead spray chamber (not cooled) and one-piece torch (1.5 mm ID
injector); Cones Nickel, Xi Design; Detector Simultaneous
pulse/analogue; Uptake Time 25 s at 50 rpm; Stabilization Delay 10 s at
17 rpm; Wash Time 40 s at 50 rpm, Survey Runs 1—scanning; Main Runs
3—peak jumping; Number of Points per Peak 1; Dwell Time/Point 5—50 ms;
Number of Sweeps/Replicate 25. Internal Standardization Technique
Interpolation, using 6Li, 45Sc, 115In, 159 Tb. Total Time per Sample
2:45 min.
Toenail
and hair samples from Al Basrah were prepared and analyzed as follows:
All reagents used were analytical grade or better. All aqueous solutions
were prepared using deionised water (18.2 MΩ Millipore, UK). A
multi-element standard and single element standard for the internal
standards (SPEX CertiPrep, UK) were used as calibration standard and
internal standard respectively for ICP-MS analysis. Concentrated nitric
acid (HNO3) and 30% v/v hydrogen peroxide (H2O2), (BDH Aristar,UK) were
used for the dissolution of samples. Analyses of toenail and hair
samples were conducted at the Inorganic Geochemistry laboratories of the
British Geological Survey (Nottingham, UK). Toenail and hair samples
were washed thoroughly following a slightly modified version of the
protocol described by Button et al. (2009), which is comparable to several published methods (Slotnick et al. 2007).
Visible exogenous material was firstly removed using plastic forceps
and a clean quartz fragment. Samples were then placed in clean glass
vials and sonicated for 5 min using 3 mL of acetone, rinsed first with
2 mL of deionised water then 2 mL of acetone, sonicated for 10 min in
3 mL of deionised water then twice rinsed with 3 mL of deionised water,
ensuring complete submersion of the sample during each step. The final
rinse solution (3 mL) was retained for immediate analysis by ICP-MS to
ensure removal of exogenous contamination was complete. The supernatants
from each step of the washing procedure were combined and reduced to
dryness in PFA vials (Savillex, USA) on a graphite hot block at 80°C.
The residue was then reconstituted in 3 mL of 1% HNO3 for analysis by
ICP-MS. After washing, toenails were left to dry at room temperature in a
clean laminar flow hood. Certified reference materials GBW 07601 human
hair and1 and NCS ZC 81002b human hair (NCS Beijing, China) were used throughout.
Both the digestion and analytical method follow the procedure described in Button et al. (2009).
Toenail samples were acid digested for total elemental determination
using a closed vessel microwave assisted digestion (MARS 5, CEM
Corporation, UK). Into each vessel 4 mL of HNO3 and 1 mL of H2O2 was
added to accurately weighed toenail and hair samples and left to stand
for 30 min before sealing the vessels. The microwave heating program
was: 100% power (1,200 W), 5 min ramp to 100°C, held for 2 min, ramped
for 5 min to 200°C then held for 30 min. The pressure in the system was
approximately 200 psi under these conditions. This method resulted in
complete sample dissolution. The solutions were transferred with MQ
water to PFA vials and evaporated to dryness on a hotplate at 110°C.
Samples were reconstituted with 1 mL of 3% v/v HNO3, heated at 50°C for
10 min and then made up to 3 mL with deionised water to give a final
solution of 1% HNO3 for direct determination via ICP-MS.
The
enamel and coronal dentine components of teeth were density separated
using a heavy liquid method. This was achieved by lightly crushing the
decoronated tooth material and adding the powder to the heavy liquid
bromoform (CHBr3) in a separating funnel. The bromoform was then slowly
diluted with acetone to achieve the optimal density for enamel and
dentine separation (2.7 g/mL). The enamel formed sediment at the base of
the funnel and was removed. The two components were washed with acetone
and dried in a laminar flow hood prior to dissolution. The teeth
samples were accurately weighed into acid washed polypropylene
autosampler tubes (Sarstedt), to which 0.2 mL of 2:1 HNO3:HCl was added,
allowed to dissolve over 5 min, then 0.8 mL of deionised water added,
left to stand for 10 min and then made up to a final volume of 10 mL
with deionised water. The sample solutions were diluted approximately
×4,000 prior to analysis to ensure a final calcium concentration of
100–200 mg L−1, to avoid matrix interference and clogging of the ICP cones and torch. The final matrix prior to analysis contained 1 HNO3
and 0.5% HCl. Multielement analysis of toenail, hair and teeth digests
was performed by inductively coupled plasma mass spectrometry (ICP-MS,
Agilent 7500, Agilent Technologies, UK). The instrument was fitted with a
micro flow concentric nebuliser and quartz Scott-type spray chamber.
The instrument response was optimised daily using a commercially
available Tune solution (SpexCertiprep). Multielement analysis was
performed in collision cell mode using He (4 L/min) to minimise
potential interferences such as that of the polyatomic ion 40Ar + 35Cl on 75As.
An internal standard comprising of Sc, Ge, Rh, In, Te, and Ir was added
to the sample line via a T-piece to monitor instrument signal
stability. The limit of detection (LOD) for the method expressed as the
mean blank signal + 3SD was as follows: Al and Fe < 2 mg/kg; V, As,
Se, Mo, Cd < 0.01 mg/kg; Mn and Zn < 0.2 mg/kg; Co, Th and
U < 0.005 mg/kg; Cr, Ni, Cu, W, and Pb were <0.07, <0.02,
<0.1, <0.08, <0.03 mg/kg respectively. Recoveries for both
reference materials were generally better than 100 ± 15% for when
compared to available reference values. SPSS version 19 was used for all
statistical analyses; an independent sample T Test was used to compare metals between two groups. A paired sample two tailed T Test was used to compare birth defects in Al Basrah data. Significance level was set at α = 0.05.
Results and Discussion
It
is well-known that exposure to stressors alters the in utero
development of a human fetus and has adverse health consequences for the
offspring, including a short gestation period, reduced birth weight,
increased risk of metabolic, cardiac and psychiatric disease, and
overall reduced lifespan (Seckl 1998; Landrigan et al. 2004; Perera et al. 2004; Llop et al. 2010).
Populations caught in war-zones or forced to live with severe
nutritional restrictions (such as those imposed on the Iraqi population
by U.N. sanctions from 1991 to 2003) suffer immediate and chronic stress
that leads to long-lasting physical and mental damage. In addition to
the harsh effects of sanctions, many Iraqi cities have experienced
large-scale bombardment. An accurate tally of the types and volume of
ammunition dropped on the Iraqi population is not available. However,
reports have indicated that large numbers of bullets have been expended
into the Iraqi environment (Buncombe 2011).
Thus the environmental contamination of Iraqi cities with materials
contained in bullets and bombs may be expected. Toxic metals such as
mercury (Hg) and Pb are an integral part of war ammunition and are
extensively used in the making of bullets and bombs (Departments of the
Army, the Navy, the Air Force, Joint Technical Bulletin 1998; US Department of the Army Technical Manual 1990).
The
case study of 56 Fallujah families and the metal analysis of hair
samples from this population indicated public contamination with two
well-known neurotoxic metals, Pb and Hg. Hair metal data from Fallujah
showed Pb to be five times higher in the hair samples of children with
birth defects (n = 44; mean ± SD 56,434 ± 217,705 μg/kg) than in the
hair of normal children (n = 11; 11,277 ± 27,781 μg/kg). Mercury was six
times higher (n = 44; 8,282 ± 25,844 μg/kg Vs n = 11;
1,414 ± 3,853 μg/kg) (Fig. 3).
Fallujah mothers who participated in this study did not take any
medication and described their diet as "good" during pregnancy. Only one
couple was first cousins. Mothers did not drink or smoke during
pregnancy. All families consumed water from local aqueducts or locally
bottled waters. Siblings of the parents had no history of children with
congenital defects. Figure 1
shows a chronological increase in the percentages of birth defects and
miscarriages in these Fallujah families. Six photographs of Fallujah
children and their conditions are provided in Fig. 2.
Mercury and Pb, two toxic metals readily used in the manufacture of
present-day bullets and other ammunition, were 6 and 5 times higher in
hair samples from Fallujah children with birth defects compared to
Fallujah children who appeared normal (Fig. 3).
Uranium, Hg and Pb, (μg/kg, mean ± SD) in the hair samples of parents
from Italy, Iran, and Fallujah (Iraq), are shown in Fig. 4.
Though statistically not significant, the hair of parents of children
with birth defects had more uranium, Pb and Hg than the hair of parents
of normal children.
Mercury and lead, (μg/kg, mean ± SD) in hair samples from Fallujah children compared to children from Italy, Palestine, and Iran
Photos of some birth defects reported from Fallujah General Hospital between May and August 2010. a Gastroschisis, b Hydrocephalus, c Encephalocele, d Macrocephaly, e Spina Bifida; f Cleft lip and palate
Uranium, mercury and lead, (μg/kg, mean ± SD) in hair samples from parents from Italy, Iran, and Fallujah Iraq
The
most common abnormalities in Fallujah children were congenital heart
defects (n = 24 out of 46), neural tube defects (n = 18 out of 46), and
cleft lip/palate (n = 4 out of 46). Cardiac defects, neural tube
defects, and facial clefting are known as folate-dependent birth defects
since folate intake reduces their occurrence (MRC Vitamin Study
Research Group 1991; Wilson et al. 2003; Obican et al. 2010).
The Fallujah study has highlighted the role of metals in the
manifestation of the current birth defect epidemic in that city. Recent
data has linked metal exposure to oxidative stress and folate deficiency
in humans (Wang et al. 2012).
We also know that in utero metal exposure can culminate in birth
deformities by increasing oxidative stress in the womb as the fetus
grows (Apostoli and Catalani 2011).
In
general, reports of health problems in the Iraqi population and in the
surrounding countries have continued to surface (Rajab et al. 2000).
News of increases in childhood cancers, of perinatal and infant
morbidity and mortality, and of unusual increases in congenital birth
defects, have continued to emerge from across Iraq. Data from a central
Iraqi city, Al-Ramadi, have corroborated the Fallujah findings (Al-Ani
et al. 2010).
Another Iraqi city where birth defects and cancers continue to climb is Al Basrah.
The
earliest data on the occurrence of congenital birth defects in Al
Basrah came from an article entitled "Incidence of Congenital Fetal
Anomaly in Al Basrah Maternity Hospital" (Alsabbak et al. 1997).
This research reported on the total number of live births (10,015) in
Al Basrah Maternity Hospital between October 1994 and October 1995. The
number of birth defects per 1,000 live births during this period was
1.37. Table 1
contains the yearly account of the number of birth defects per 1,000
live births in Al Basrah Maternity Hospital from 2003 to 2011. Central
nervous system related defects occurred most frequently. Statistical
analysis of this data has shown no significant difference between the
number of children born with anencephaly and the number born with Spina
Bifida (p = 0.28). There were significantly more cases of
anencephaly than of hydrocephalus, limb deformity, omphalocele, or short
extremities (p = 0.009, p = 0.005, p = 0.000, p = 0.000).
In addition, the number of Spina Bifida cases was significantly higher
than the number of hydrocephalus, limb deformity, omphalocele, or short
extremity cases (p = 0.05, p = 0.005, p = 0.000, p = 0.001).
Within 8 years, the occurrence of congenital birth defects in Al Basrah
Maternity Hospital increased by an astonishing 17-fold.
The prevalence of congenital hydrocephalus in California (US) has been reported as 0.6 per 1,000 (Jeng et al. 2011).
Worldwide hydrocephalus affects about one in every 1,000 live births.
The reported numbers of hydrocephalus from Al Basrah Maternity Hospital
were 3.5 times higher than the world average and six times higher than
in the United States. Defects of the abdominal wall, like omphalocele
and gastroschisis, were also frequently reported in Al Basrah.
Omphalocele generally occurs in 0.25/1,000 live births and is associated
with a high rate of mortality and severe malformation, such as cardiac
anomalies and neural tube defects. The average number of omphalocele
observed in Al Basrah Maternity Hospital between 2003 and 2011 was
3.3/1,000 live births.
Neural
tube defects (NTDs) occur very early in human development. The
prevalence of NTDs in the mainland United States is 1/1,000 live births
(CDC; Williams et al. 2002). Some of the highest numbers of NTDs have been reported from coal mining regions in China (10/1,000) (Li et al. 2006).
The occurrence of NTDs in Al Basrah (12/1,000) is the highest ever
reported and it is increasing. Our data has shown that in Al Basrah, the
total number of birth defects more than doubled between 2003 and 2009.
A
comparison between the metal levels in the hair (n = 6) and toenail
(n = 7) of parents of children with birth defects from Al Basrah, and
the associated p values, has been presented in Table 2.
For most metals (Al, Mn, Co, Cu, Zn, Mo, Pb, Th, and U), hair contained
significantly higher amounts of the metal than did toenail, suggesting
that hair is a better biomarker of exposure. Examining the Pb hair
levels of parents from Al Basrah and Fallujah revealed that the hair of
parents of children with birth defects in Al Basrah had 6,500 ± 8,589
(μg/kg); in Fallujah this value was 3,950 ± 3,133 μg/kg; both values
being considerably higher than Pb found in the hair samples of parents
of normal children from Fallujah (2,012 ± 2,052 μg/kg). The 1.6-fold
higher Pb in the Al Basrah parents’ hair compared to Fallujah parents’
hair may be explained by the fact that Al Basrah is an oil-industry
dominated area whereas Fallujah is not. Overall, parents of children
with birth defects from Al Basrah and Fallujah had twofold, and
one-fold, higher Pb in their hair than did parents of normal children
respectively. Al Basrah parents who had children with birth defects also
had 1.4 times higher enamel Pb (n = 5, 2,497 ± 1,400) than did parents
of normal children (n = 5, 1,826 ± 1,619). Additional samples of teeth
from the parents of children with and without birth defects from Al
Basrah are necessary to help draw reliable statistical conclusions for
this population.
Comparison of metal levels in hair verses toenail from parents of children with birth defects from Al Basrah, Iraq
Table 3
contains a literature review of the metal levels in whole deciduous
teeth of children from different geographical locations. Enamel is a
hard and dense material which is formed during fetal life and it
receives small amounts of systemic blood flow thereafter. For this
reason it is considered to primarily reflect prenatal exposure to
metals. Whole-tooth metal analysis would include enamel, dentin,
cementum, and dental pulp. The impact of living in a large city with
dangerous levels of air pollution is evident in the high levels of Pb in
teeth from Mexico City and Karachi. Similarly, the level of Pb in teeth
from Canadian mining areas is indicative of an exposed or impacted
population. Mean whole tooth Pb reported from other locations was
1.5 μg/g. Hence, the tooth Pb level of an unimpacted population is more
accurately estimated by this 1.5 μg/g value. The enamel portion of the
deciduous tooth from a child with birth defects from Al Basrah
(4.19 μg/g) had nearly three times higher Pb than the calculated value
for a whole tooth from an unimpacted population. Additional samples of
deciduous teeth from Al Basrah children with birth defects are necessary
to help draw reliable statistical conclusions for this population.
Samples of deciduous teeth are currently being collected from impacted
(Al Basrah, Iraq) and unimpacted (Isfahan, Iran) areas to enable us to
draw meaningful conclusions in this regard. Interestingly, mining,
smelting, and living near industry or hazardous waste sites have all
been associated with an increased risk of birth defects (Ahern et al. 2011; Zheng et al. 2012; Suarez et al. 2007).
A
literature review of selected metal levels in the whole deciduous teeth
of children from various geographical locations verses the sample from
Al Basrah, Iraq
Present
knowledge on the effects of prenatal exposure to metals, combined with
our results, suggests that the bombardment of Al Basrah and Fallujah may
have exacerbated public exposure to metals, possibly culminating in the
current epidemic of birth defects. Large-scale epidemiological studies
are necessary to identify at-risk populations in Iraq. The recognition
that birth defects reported from Iraq are mainly folate-dependent offers
possible treatment options to protect at-risk populations.
Acknowledgments
We
thank Drs. Hossein Malekafzali, Howard Hu, and Timothy Johnson for
their interest in this research. We thank Drs. Vasantha Padmanabhan,
Marjorie Treadwell, Kataneh Salari, for their critical reading of this
manuscript, and we thank Blaine Coleman for technical assistance.
Funding for this research was provided by the University of Michigan
Department of Obstetrics and Gynecology.
This
article is distributed under the terms of the Creative Commons
Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
References
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2012/10/11
Democracy: metal contamination and the epidemic of congenital birth defects in Iraqi Cities
