Environmental Toxicology and Pharmacology 85 (2021) 103629

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Environmental Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/etap

Lead concentrations in breast milk of Moroccan nursing mothers and

associated factors of exposure: CONTAMILK STUDY
Abha Cherkani-Hassani a, b, *, Meriem Slaoui c, Imane Ghanname d, e, Brahim Mojemmi b,
Rachid Eljaoudi f, Abdelilah Belhaj g, Brahim Kers g, Majda Flayou g, Nezha Mouane a, h
a
Unit of training and research in Nutrition and Food Science. Faculty of Medicine and Pharmacy, University Mohammed V, Rabat, Morocco
b
Laboratory of analytical chemistry and food science, Faculty of Medicine and Pharmacy, University Mohammed V, Rabat, Morocco
c
University Mohammed V, Rabat, Morocco
d
Team research of Pharmacoeconomics and Pharmacoepidemiology, Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy, University
Mohammed V, Morocco
e
Faculty of health sciences, International University of Casablanca, Morocco
f
Laboratory of pharmacology and toxicology, Faculty of Medicine and Pharmacy, University Mohammed V, Rabat, Morocco
g
Laboratory of Quality Control of Waters, National Office of Electricity and Drinking Water (ONEE), Rabat, Morocco
h
Department of Pediatric, Hepatology, Gastroenterology and Nutrition PIII-Children’s Hospital, Rabat, Morocco

A R T I C L E I N F O A B S T R A C T

Edited by M.D. Coleman The aim of CONTAMILK study was to estimate levels of contamination of breast milk (BM) of Moroccan nursing
mothers by some xenobiotics including, lead (Pb), to identify associated factors of exposure and to determine the
Keywords: daily intake of newborns. Lead concentrations were determined in 70 samples of colostrum by ICPMS and a
Lead levels structured questionnaire was filled during milk collection to report participants’ data. The median lead con­
Breast milk
centration was 908 μg/L (range 1.38–515,39 μg/L) and in 79 % of samples, levels were higher than the normal
Associated factors
range reported by the World Health Organization (WHO) in BM (2− 5 μg/L). Indeed, preterm delivery, frequency
Daily intake
Morocco of use of cosmetic powders and lipsticks were significantly associated with the level of lead in BM. The estimated
daily intake was greater than the tolerable daily intake (TDI) of the European food safety authority (0.5 μg/kg/
day) for 39 babies and 6 babies according to the WHO (3.6 μg/kg/day).

1. Introduction First, we conducted and published a systematic review about lead in

Many authors have considered breast milk as a relevant and rec­
ommended biological matrix for biomonitoring studies on toxic envi­
ronmental contaminants, because it may serve as a biomarker of both
maternal and fetal exposure to a few environmental chemicals (Ander­
son and Wolff, 2000; LaKind et al., 2002; Needham and Wang, 2002).
Lead (Pb) is a widespread and toxic heavy metal, which can be
transferred from the body reserves and the blood into the breast milk
(BM) of exposed mothers. Lead exposure affects the central nervous
system of infants and children, while the cardiovascular system and
kidneys are the most affected in adults (ATSDR, 1993; Sanders et al.,
2009; Wani et al., 2015). Lead compounds are classified as probably
carcinogenic to humans (Group 2A) in 2006 (IARC, 2006). The normal
range of lead in breast milk reported by the World Health Organization
(WHO) is 2− 5 μg/L (WHO, 1989).
BM before this study to explore the literature. Many studies around the
world reported high contamination of BM by lead. The levels were
influenced by several factors, such as the place of residence of the
mothers, maternal age, maternal dietary intakes, stage of lactation,
smoking habits and parity (Cherkani-Hassani et al., 2020a).
The objective of the present survey is to report lead levels in breast
milk of lactating Moroccan women and its association with several
maternal parameters and habits, also to estimate the daily intake of the
newborns.
2. Material and methods
2.1. Study design and population
CONTAMILK study was conducted at Souissi maternity hospital of

* Corresponding author at: Faculty of Medicine and Pharmacy, University Mohammed V, Rabat, Morocco.
E-mail address: abha.cher@gmail.com (A. Cherkani-Hassani).

https://doi.org/10.1016/j.etap.2021.103629
Received 14 September 2020; Received in revised form 1 March 2021; Accepted 3 March 2021
Available online 5 March 2021
1382-6689/© 2021 Elsevier B.V. All rights reserved.
A. Cherkani-Hassani et al.
Rabat, to investigate levels of contamination of human breast milk by
some toxic chemicals including lead among nursing mothers from Rabat
and its regions in Morocco. Thus, 70 breast milk samples were collected
between February and December 2015 from lactating Moroccan women
living in Rabat and its regions, and who voluntarily participate in this
cross-sectional study.
The inclusion criteria were healthy women who gave birth to healthy
newborns with normal birth weight and normal clinical examination at
sampling time-point. The exclusion criteria were chronic diseases (dia­
betes, gestational hypertension, asthma, etc.), fever, infections, meta­
bolic diseases, diseases of breast or central nervous system,
malnutrition, and maternal allergy.
2.2. Sampling procedure
The areola and nipple area were thoroughly washed with deionized
water, and about 10 mL of breast milk (one sample) were obtained from
the lactating women between the 2nd and 5th days post-delivery in the
morning. The colostrum samples were collected by a manual milk pump
and were kept in acid prewashed plastic containers, then they were
transported on an ice cooler and stored at − 70 ◦ C until analysis.
2.3. Questionnaire
A detailed questionnaire was administered by the same interviewer,
during the collection of milk to report data concerning socio-
demographic and economic data (place of residence, age, working sta­
tus of the women and the husband income, level of education),
anthropometric data (weight before and during pregnancy and height of
the mother also, gender and birth weight of the newborn, etc), clinical
data: health status, parity, gestational age, parturition, gyneco-obstetric
history, miscarriages, anemia history and status, current medication,
supplementation, lactation history, amalgam teeth fillings…and other
personal habits (smoking habit or exposure and cosmetics, etc)
Some data were taken from the report of the maternity hospital or/
and from the maternal health record. The gained weight during preg­
nancy was calculated for each woman.
The questions on diet included vegetables, fruits, fish, shellfish,
meat, grains products, canned food, soft drinks (with the type of pack­
aging), etc. whereby 4 categories, namely: never, occasionally (≤ once a
week), frequently (≥ twice a week) and every day, were provided to
specify consumption habits and to identify potential sources of toxic
metal intake. More other details were recorded throughout the
interview.
Further details about the questionnaire, the dietary habits, and the
frequency of consumption of foodstuffs were reported in our previous
study (Cherkani-Hassani et al., 2020b).
2.4. Ethical approval
The study protocol was previously approved by the Ethics Committee
of Biomedical research (CERB) of the Faculty of Medicine and Pharmacy
of Rabat (N◦ 700/18− 07-2014). All mothers were informed about the
purpose of the study and signed informed consent before the collection
of breast milk.
2.5. Analytical methods
2.5.1. Sample pretreatment and mineralization
Before analysis, breast milk samples were thawed and homogenized
in a warm bath, then in closed Teflon containers, an aliquot of 1 mL of
milk the sample was reacted with 5 mL of nitric acid (65 % Suprapur,
FLUKA, Germany) and 1 mL of hydrogen peroxide H2O2 (30 % Sigma-
Aldrich, Germany) and were closed and remained for 1 h at room tem­
perature. Next, samples were mineralized in the microwave digestion
system (Anton Paar Multiwave 3000) (for 30 min at 121 ◦ C; 800 W)
2
Environmental Toxicology and Pharmacology 85 (2021) 103629
according to the manufacturer’s application notes.
With each series of 12 samples, together one blank (HNO3 and H2O2)
and one sample of certified reference material ERM BD-150 (skimmed
milk powder, Institute for Reference Materials and Measurements, Geel,
Belgium) were treated as described for other milk samples.
Afterward, the clear, colorless digest was quantitatively transferred
to a polypropylene bottle and adjusted to 10 mL with deionized water
(>18 MΩ).
The Certified Reference Material spiked skim milk powder ERM BD-
150 (Institute of Reference Materials and Measurements, Geel, Belgium)
was used as control and treated as described for other milk samples.
All lab-ware was acid washed before use by soaking in 10 % HNO3
overnight and thoroughly rinsed with deionized water.
2.5.2. Calibration and lead analysis
Lead analysis was performed by inductively coupled plasma-mass
spectrometry (ICP-MS; THERMO SCIENTIFIC XSERIES 2) with an auto
sampler Cetac 500. Analytical standards used for calibration were pre­
pared from single analytical standard solutions of lead (1 g/l, CPA
chem). The calibration curve was plotted with standard solutions of lead
(0− 0.2-1− 2-10− 20-100− 200 μg/l).
2.5.3. Quality control
The accuracy of the analysis was checked by various methods
including sampling chemical blanks and reference material (ERM BD
150). The observed lead concentrations were: 0.019 ± 0.006 mg/kg, (n
= 6) were in agreement with the certified values of 0.019 ± 0.004. The
mean recovery rate of lead from the reference materials was 102 %.
Further, the metal content of each sample was obtained from the mean
value of 3 replicate determinations.
Calibration curves were regularly checked during analyses for line­
arity (r2 ≥ 0.99), the correlation coefficient (R) of the calibration curve
was R2 = 0.999958. The LOD was 0.255 μg/l, calculated as three times
the standard deviation (SD) based on 10 blanks samples values, which
were included in the analytical run, and the LOQ was 0.85 μg/L,
calculated as 10 times the standard deviation (SD).
2.6. The daily intake
The exposure of infants to lead was estimated based on its concen­
tration of lead in BM, average daily consumption of BM of the newborns,
and birth weight.
The estimated daily intake (EDI) by the infant was estimated ac­
cording to the following equation (Chien et al., 2006).
EDI Pb (μg/kg of body weight/day) = VBM X C Pb / BW
With: VBM : average volume of BM consumed by the newborn (L) per day
;
CPb : Concentration of lead in BM (μg/L) ;
BW: newborn birth weight (kg)
The average volume of BM consumed by a newborn is 40− 50 ml on
the 1st day, 100 mL on the 2nd day, 150 mL on the 3rd day, 200 mL on 4th
day, and 250 mL on the 5th day (Turconi et al., 2004).
The average day of BM collection in the present study is the 4th day,
therefore the average volume of BM which will be considered is 200 mL.
2.7. Statistical analysis of the data
Statistical analysis of the data was performed using both Microsoft
Office Excel (Microsoft Corporation, Redmond, WA) and the Statistical
Package for Social Sciences (SPSS) software package, version 13.0 for
Windows. Statistical methods were used to calculate descriptive statis­
tical parameters (mean, median, standard deviation, minimum and
maximum) for each element. Comparison of average values between
groups was calculated using Student’s t-test. When the examined
A. Cherkani-Hassani et al. Environmental Toxicology and Pharmacology 85 (2021) 103629

variables did not have a normal distribution, the variable intergroup Table 1
comparison was performed with the following non-parametric tests: Anthropometric. Socio-demographic and Economic characteristics of the study
Mann-Whitney for comparison between two groups; Kruskal-Wallis test population.
for comparison among three or more groups. The confidence level was All participant (70)
set at 95 %. We used simple and multiple linear regression to investigate Mothers/newborns Number Mean ± SD Median Range
the association between the studied parameters and lead concentrations. (%)

Age (Years) 29.31 ± 28.00 [19 ; 44]

3. Results 6.74
Gestational gain weight 10.59 ± 10.00 [0 ; 22]
3.1. Population’s characteristics’ (Kg) 4.74
Height (cm) 159.74 ± 160.00 [150− 171]
5.37
Table 1 reports the general description of the participants. The pre­ BMI
sent study included 70 healthy women with a mean age of 29.3 ± 6.74 <18.5 1 (1%)
years and ranged between 19 and 44 years. Participants’ newborns were 18.5 - 24− 99 37 (54 %)
males in 43 women (61.4 %) and females in 25 women (35.7 %), while 25 - 29.99 20 (29 %)
4 (6%)
twins were delivered by only two women (2.9 %). Among our volunteer ≥30
Maternal education :
women, 56 (80 %) were from urban area. Concerning education, only Illiterate 23 (32.9
41.4 % had middle and high school level while 23 women (32.9 %) were %)
illiterate. The occupation status shows that almost all of the women Elementary school 15 (21.4
(82.9 %) were stay-at-home mothers. We found that the family income %)
Middle and High school 29 (41.4
was low (<3500 MAD ~ 340$) in 71.4 % of the studied population. %)
Maternity characteristics were as follows: 39 women (55.7 %) University degree 3 (4.3 %)
delivered normally and 31 women (44.3 %) were primiparous. History Total monthly family
of miscarriages was noted in 15 women (21.4 %) and hormonal income
50 (71.4
contraception history was reported in 36 women (51.4 %). Previous <3500
%)
breastfeeding was reported by 53 % of the women. 3500− 5299 11 (15.7
On the other hand, anemia history was reported in only 5 women %)
(7.1 %) while 36 mothers noticed to have anemia during pregnancy and 5300− 9999 7 (10 %)
60 women (85.7 %) took vitamins and minerals supplements during <10,000 2 (2.9 %)
Mode of Delivery :
pregnancy. Normal 39 (55.7
%)
3.2. Lead concentration in breast milk Cesarean 31 (44.3
%)
Gestational duration 39.49 ± 39.50 [36 ; 42]
The lead concentration in BM samples was not normally distributed,
1.38
so the median was 9.08 μg/L. the highest concentration was 515.39 μg/ Preterm 18 (25.7
L. In 55 samples (78.5 %), lead levels were higher than the normal range %)
reported by the World Health Organization (WHO) in breast milk (2− 5 Full term 52 (74.3
μg/L) (Table 2). %)
Parity :
Primiparous 31 (44.3
3.3. Association of some factors on lead levels in breast milk %)
Multiparous 39 (55.7
3.3.1. Results of non-parametric tests %)
Average duration of
Gestational duration affects significantly the level of lead in BM (p =
previous of breast
0.031). Mothers who delivered before term (<39 weeks of amenor­ feeding
rhoea) had more lead in their milk than those who delivered at term Never 33 (47.1
(≥39 weeks of amenorrhoea) :13.97 vs 7.71μg/l. %)
In addition, the frequent use of cosmetic powders was significantly 3− 6 months 8 (11.4
%)
associated with lead in BM (p = 0.009). Indeed, the frequent use of 7− 12 months 9 (12.9
lipsticks, kohl, and lightening creams appeared to affect lead concen­ %)
trations (p < 0.05). 13− 18 months 6 (8.6 %)
On the other hand, it appears that a high frequency of consumption 19− 24 months 14 (20 %)
Mothers with history of
of legumes, and dried fruits tended to increase the concentration of lead
miscarriages
in BM; similarly, mothers who drank tap water had more lead in their Yes 15 (21.4
milk than those who drank well water. Conversely, high consumption of %)
tea, chicken, and canned foods appeared to decrease the level of lead in No 55 (78.6
BM, but differences were not statistically significant. Tables 1 and 2 in %)
Hormonal contraception
the supplemental material present the results of non-parametric tests. history
Yes 36 (51.4
3.3.2. Results of simple and multiple regression %)
The results of the simple linear regression revealed that the con­ No 34 (48.6
%)
centration of lead in BM were significantly associated with gestational
Anemia history
age (β = -0.323, p = 0.006) and the frequency of lipstick use (β = 0.24, p Yes 5 (7.1 %)
= 0.046). However, some associations very close to the significance no 65 (92.9
were observed such as the frequency of tea consumption (β = -0.234, p = %)
0.052) and the presence or absence of dental amalgam (β = -0.355, p = Anemia in pregnancy

0.064). However, multiple linear regression did not show any significant (continued on next page)

3
A. Cherkani-Hassani et al. Environmental Toxicology and Pharmacology 85 (2021) 103629

Table 1 (continued ) day). Additionally, it was greater than the tolerable daily intake (TDI)
All participant (70) sets by the WHO (3.6 μg/kg/day) (WHO/JEFCA, 2010) for 6 babies and
Mothers/newborns
EFSA (0.5 μg/kg/day) (EFSA EFSA, 2012) for 39 babies.
Number Mean ± SD Median Range
(%)
Table 3 summarized the mean, standard deviation, median, inter­
quartile range, and minimum and maximum values of the estimated
Yes 36 (51.4
daily intake (EDI) of lead by the newborns in μg per kg of body weight
%)
No 34 (48.6 per day.
%)
Supplementation during 4. Discussion
pregnancy
Yes 60 (85.7
%)
4.1. Levels of lead in breast milk
No 10 (14.3
%) Our results showed high lead concentrations in the majority of
Past or Current smoking samples, and 55 samples (78.5 %) exceeded the maximum tolerated
habits
limit in human milk (5 μg /L), the median of the concentrations was 9.08
Yes 0
No 100 (100 μg / L.
%) Indeed, two Moroccan studies have already evaluated the level of
Husband or family smoke lead in breast milk in polluted areas of the city of Marrakech and the
at home results were high. The mean concentration of lead in the first study was
Yes 29 (41.4
%)
58 μg / L and in the second study was 36.3 ± 26.1 μg / L (Lekouch, 2004;
No 41 (58.6 Souad et al., 2006). However, our study is the first study conducted in
%) Morocco investigating the impact of several maternal factors on the level
Newborn gender : of lead in breast milk, especially the colostrum of non-exposed Moroccan
Female 25 (35.7
breastfeeding mothers.
%)
Male 43 (61.4 Previous studies from Morocco assessed lead in blood, and reported
%) high levels too: 86.9 ± 42.1 μg/L and 35.8 ± 34.15 μg/L in Rabat
Twins* 2 (2.9 %) (Khassouani et al., 1997; Shaimi et al., 2014), as well, 53.74 ± 42.08 in
Newborn weight# 3400.14 ± 3425.00 [2050 ; Casablanca, 50.4 ± 27.5 in Settat and 40 μg/L in Fez (El Kettani et al.,
562.83 4800]
< 2500g 5 (7.1 %)
2010; Pawlas et al., 2013; Shaimi et al., 2014).
2500− 3000 8 (11.4 Several studies around the world had also found very high concen­
%) trations of lead in colostrum and which exceeded the normal range of
3000− 4000 49 (70 %) the WHO, except studies from Austria, Croatia, Greece, Portugal, and
8 (11.4
<4000
Slovakia (Almeida et al., 2008; Krachler et al., 1998; Leotsinidis et al.,
%)
2005; LetiniĿ et al., 2016; Ursinyova and Masanova, 2005). Table 4
*
2 women gave birth to twins (male and female). summarizes the main results of those studies.
#
the mean weight of the twins were considered. However, for mature and transitory milk, levels of lead in the Med­
iterranean countries were different from one country to another. The
median concentrations were: 16.64 μg/L in Spain (Garcia-Esquinas
Table 2
Lead concentrations in breast milk of Moroccan nursing mothers (n = 70).
et al., 2011), 20.59 μg / L in Turkey (Orun et al., 2011), 7.75 μg / L in
Italy (Turconi et al., 2004) and 4 μg / L in Palestine (Shawahna et al.,
Mean ± Median Range ≤2 2− 5 5− 20 ≥ 20
2016). While in Egypt and Greece 90 ± 29 μg / L and 30.6 μg / L
SD μg/L μg/L μg/L μg/L
respectively (average) (Saleh et al., 1996; Vavilis et al., 1997).
23.08 ± 9.08 [1.38;515.39] 2(2.9 13 43(61.4 12 Other studies in Italy and Greece reported much lower mean levels:
62.75 %) (18.6 %) (17.1
%) %)
0.849 μg / L and 0.48 ± 0.6 μg / L, respectively (Abballe et al., 2008;
Leotsinidis et al., 2005). In Europe, too, lead in human milk was low:
1.55 ± 1.38 μg / L in Portugal, 4.7 μg / L in Slovakia and 0.73 ± 0.70 μg /
association between lead concentration in BM and the studied factors. L in Austria, while in Poland, the average concentration was higher:
Table 3 in the supplemental material presents the results of simple and 6.331 ± 4.614 μg / L (Almeida et al., 2008; Gulson et al., 1998a; Ursi­
multiple regression. nyova and Masanova, 2005; Winiarska-Mieczan, 2014).
On the other hand, studies conducted in the Middle East reported
3.4. Estimation of daily intake of lead very high levels of lead in BM, the mean concentrations measured were:
25.9 ± 18.40 μg / L in Iraq, 10.39 ± 4, 72 μg/l in Iran and 31.67 ± 45.66
The estimated daily intake (EDI) of lead (μg per kg of body weight μg/L in Saudi Arabia (Al-Saleh et al., 2003; Nassir et al., 2012; Rahimi
per day) following infants’ consumption of 200 mL of breast milk varied et al., 2009). Nevertheless, in the Arab Emirate, the concentration was
between 0.08 and a maximum of 33.69 μg/kg/ day (median 0.54 μg/kg/ much lower: 0.019 ± 0.055 μg / L (Abdulrazzaq et al., 2008).

Table 3 4.2. Factors associated with lead levels in breast milk

Estimation of daily intake (EDI) of lead (μg/kg/day) after consumption of 200
mL of breast milk by the 70 newborns. Many researchers have explored the factors affecting the levels of
EDI of lead (μg/kg/day) TDI WHO/ TDI EFSA (0.5 lead contamination in human milk; however, the results were very
JECFA (3.6 μg/ μg/kg/day) controversial among the studies.The present report also revealed that
kg/day) the lead level increases significantly in BM of women who delivered
Mean ± Median Percentile Min- EDI < 3.6 μg/ EDI<0.5 μg/ before term which coincides with the finding of Gundacker et al.
SD Max kg/day kg/day (Gundacker et al., 2002).
1.42 ± 0.54 0.39 ; 0.08- 6 (8.33 %) 39 (47 %) The area of residence was widely investigated by several authors,
4.06 0.92 33.69
and the majority of studies have found significantly higher

4
A. Cherkani-Hassani et al. Environmental Toxicology and Pharmacology 85 (2021) 103629

Table 4
Worldwide lead concentrations in colostrum.
Concentrations μg/L Notes
References Country Number of samples
Mean ± SD GM* Median Range

Sternowsky and Wessolowski 1985 Germany 20 15.5 ± 6.1 Population urbaine et rurale
Ong et al. 1985 Malaysia 114 47.84 ± 12.48 24.96− 106.08
90 ± 29 Urban women
Vavillis et al. 1997 Greece 91
84 ± 24 Rurale women
26.67 ± 2.64 Norilsk city
Klopov 1997 Russian Arctic 42
22.5 ± 3.56 Salekhard city
Krachler et al., 1998 Austria 13 2.3 1
7.75
Turconi et al., 2004 Italy 143 Urban & rural population
2− 216
0.44
Leotsinidis et al., 2005 Greece 180 0.48 ± 0.6
ND-2.3
Ursinyova and Masanova, 2005 Slovakia 158 4.7 ± 4.1 3.4
8.59*±10.95 Women eating chinese herbs (n = 35)
Chien et al., 2006b Taiwan 72
6.84*±2.68 control (n = 37)
1.17
Almeida et al., 2008 Portugal 34 1.55 ± 1.38
0.06− 5.43
Chao et al., 2014 Taiwan 45 13.22 ± 3.58
8.7
Industrial zone (n = 36)
<LD-96
Hamzaoglu et al. 2014 Turkey 53
10.1
Non industrial zone (n = 17)
<LD-26.7
LetiniL et al. 2016 Croatia 20 5 Femmes non fumeuses
9.08
This study Morocco 70 23.08 ± 62.75 Urban and rural women
1.37− 515.39
*
GM: geometric mean; D: not detected; LD: limit of detection.

concentrations of lead in women living in urban and industrial areas
(Al-Saleh et al., 2003; Cinar et al., 2011; Nassir et al., 2012; Park et al.,
2017); which has been explained by the effect of industrialization
(WHO, 1989). Although, in our study, the difference was not statistically
significant which is consistent with several reports (Abballe et al., 2008;
Garcia-Esquinas et al., 2011; Orun et al., 2011; Turconi et al., 2004;
Ursinyova and Masanova, 2005).
Concerning maternal age, we found that this parameter was not
associated with lead levels in BM, which agrees with several findings
(Abdulrazzaq et al., 2008; Al-Saleh et al., 2003; Dursun et al., 2016;
Garcia-Esquinas et al., 2011; Leotsinidis et al., 2005; Matos et al., 2014;
Orun et al., 2011; Park et al., 2017). However, some authors have found
significantly higher lead concentrations in older mother’s milk (Chao
et al., 2014; Ettinger et al., 2004; Rahimi et al., 2009; Winiarska-­
Mieczan, 2014; Younes et al., 1995); which confirms the results of some
studies that have established that maternal body burden in lead is higher
in older women and is influenced by parity (Ettinger et al., 2005; Sil­
bergeld, 1991). Indeed, significant relationships between parity and
lead levels in BM have been reported by some similar studies (Chao
et al., 2014; Ettinger et al., 2004; Frković et al., 1997; Rahimi et al.,
2009; Ursinyova and Masanova, 2005). In our study, primiparous
mothers had more lead in their milk than multiparous women but the
difference was not statistically significant, which coincides with the
findings of Ursinyova and Masanova (2005).
We also noted that supplementation with vitamins and minerals
during pregnancy significantly reduced the level of lead in BM. da Silva
Anastácio et al. (2004) and Ettinger et al. (2004) reported that calcium
intakes decreased lead levels in BM.
As for dietary habits, according to the literature, consumption of the
following foods appears to affect lead levels in BM: coffee and dairy
products (Garcia-Esquinas et al., 2011), cereals (Gundacker et al., 2002),
cheese, rice & red meat (Leotsinidis et al., 2005) and fish (Al-Saleh et al.,
2003; Gundacker et al., 2002).
The present study did not show any significant associations but some
differences were observed. Women who drank tap water appeared to
have more lead in their milk than those who drank well water. This
finding is in agreement with the result of Nassir et al. (2012) who found
a significant association for this factor. In this survey, we also observed
that high consumption of legumes appears to increase lead in BM, while
low consumption of tea and poultry appears to decrease lead levels in
BM.
Some local habits, like the use of pottery in cooking, did not affect
lead levels BM samples of the nursing mothers. However, a Moroccan
study assessed the level of lead in pottery utensils such as tagines (a
traditional earthenware pot) and reported that some samples were
highly contaminated by lead exceeding the Moroccan limit (2 mg/L)
(Gouitaa 2016).
Regarding cosmetic habits, significant associations were noted be­
tween the increase of lead in BM and the frequent use of lipsticks and
cosmetic powders, which coincides with the finding of Vahidinia et al.
(2019). In addition, kohl and skin lightening creams were also linked
with increased lead exposure, which is in agreement with Shawahna
et al. (2016). Indeed, several studies confirmed the presence of lead in
many cosmetic products (Bocca et al., 2014; Siti Zulaikha et al., 2015).
Indeed, a Moroccan study analyzed several samples and textures of kohl
and found high levels of lead and which do not comply with interna­
tional standards (Gouitaa et al., 2016). In our survey, higher lead levels
in milk were observed in kohl users too but the difference was not sta­
tistically significant.
4.3. Daily intake of lead
Several studies estimated the daily or weekly intake considering the
average volume of BM consumed per day. Table 5 represents the results
reported by some studies.
The results of the present study (median 0.54 μg/kg/ day) is in
concordance with a study from Italy conducted by Turconi et al. (2004),
who found a similar daily intake of lead (0.47 μg/kg/ day) considering
the same conditions (volume of 200 mL of BM consumed per baby per
day and a mean weight of 3.3 kg for babies). Moreover, Ursinyova and
Masanova (2005) found similar values in Slovakia 0.77 μg/kg/ day
(weekly intake 5.4 μg/kg), even when the consumed milk estimation
was higher than 575 g/3.45 kg. Otherwise, Leotsinidis et al. (2005)
found much lower weekly intakes in colostrum, in Greece (milk volume:
350 mL/3.5 kg). However, Winiarska-Mieczan (2014) reported the
highest daily intake, in 2014 in Poland, which was 12 μg/kg/day
(weekly intake 84 μg/kg).

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A. Cherkani-Hassani et al. Environmental Toxicology and Pharmacology 85 (2021) 103629

Table 5
Estimated daily intake (EDI) of lead by infants during the lactation period calculated by some studies.
Country Number of newborns ; Average weight EDI of Pb μg/kg/day (Min-Max) Breast milk consumed per day Lactation stage References

Nigeria 34 ; 4.6 kg 9.9 700 mL 2− 24 Wpp Vanderjagt et al. 2001

Saudi Arabia 362 34.26* 850 mL 1er Mpp Al-saleh et al., 2003
0.094# 40 mL 1st Dpp
Italy 143 ; 3.3 kg 0.47# 200 mL 4th Dpp Turconi et al., 2004
0.70# 300 mL 6th Dpp
0.28* 100 mL/kg of bw 3rd Dpp
Greece 180 Leotsinidis et al., 2005
0.49* 150 mL/kg of bw 15th Dpp
Slovakia 158 ; 3.45 kg 5.4*(0.4− 28.5) 1/6 du bw 4ème Dpp Ursinyova and Masanova, 2005.
Taiwan 58 ; 3.09 kg <0.1− 1.8 400− 500 ml 1st Wpp Chien et al., 2006b
Poland 59 84* 1st Mpp Winyarska et al. 2014
Morocco 70 ; 3.4 kg 0.54# (0.08–33.69) 200 mL 2nd au 5th Dpp This study

Dpp:day postpartum.
*
weekly intake.
#
median; bw: body weight. Mpp: month postpartum Wpp: week postpartum.

5. Conclusion
In the present study, the majority of the samples (78.5 %) were
higher than the normal range of 2− 5 μg/L reported by the World Health
Organization (WHO), which underscores the high contamination of
breast milk of Moroccan lactating mothers by lead. However, preterm
delivery and the use of cosmetic powders and lipsticks were significantly
associated with the level of lead in BM samples. Further, the daily intake
was higher than the daily tolerable daily intake sets by the World Health
Organization (WHO) and the European Food Safety Authority (EFSA) for
some newborns.
Accordingly, awareness campaigns are needed to inform the public,
especially young and pregnant women, about adequate measures of
prevention to limit exposure to this toxic heavy metal. Also, periodic and
multicentric surveys are recommended to assess lead levels through
large cohorts.
CRediT authorship contribution statement
Abha Cherkani-Hassani: study design, samples and data collection,
analytical analysis, interpretation of results, and the writing of the
manuscript, This article is a part of the thesis of ACH. Meriem Slaoui:
statistical analysis and interpretation of results. Imane Ghanname:
statistical analysis. Brahim Mojemmi: revision of the manuscript.
Rachid Eljaoudi: revision of the manuscript. Abdelilah Belhaj:
analytical analysis. Brahim Kers: analytical analysis. Majda Flayou:
analytical analysis and interpretation of results. Nezha Mouane:
methodology and supervision of all the stages of the study.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The authors thank kindly the volunteer mothers for the donation of
milk samples and their time to answer questions and all the staff of the
Maternity Hospital of Souissi of Rabat for their collaboration by allow­
ing the collection of human milk samples. The authors are also grateful
for the technical assistance of the laboratory of quality control of waters
of the National Office of Electricity and Drinking Water (ONEE), Rabat,
Morocco.
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