Chlorinated pesticide residues in human breast milk collected from southern Jordan in 2012/2013

Abstract

Chlorinated pesticide residues in human breast milk from five southern Jordan districts were analyzed in 2012/2013. The total number of samples from all districts was 100. The number of samples gathered from each district was collected according to their population densities. The present study shows that 59% of the samples which contained pesticides’ residues were p,p’-DDE, 30% contained β-HCH, 10% contained p,p′-DDT, 6% contained α-HCH, 2% contained heptachlor and 1% contained endrin. These human milk samples were free of aldrin, dieldrin, α-endosulfan, β-endosulfan, HCB, γ-HCH, o,p′-DDD, o,p′-DDT and o,p′-DDE. The levels of the six mentioned compounds detected in all mother’s milk samples were higher in Ghor El-Safi district compared with the other four districts. In this study, it is recommended to continue the pesticides residues monitoring in all parts of Jordan, particularly in Ghor El-Safi district and other regions in the cultivated Jordan Valley.

• Breast milk
• chlorinated pesticides
• residues
• Jordan

Introduction

Agricultural activities are closely associated with intensive use of pesticides to control agricultural pests to increase the production of planted areas and to control public health insects to prevent epidemic diseases.

Human milk for infants is considered to be the natural superior food to meet their nutritional need to grow. On the other hand, human milk is suitable matrix for pollutants accumulation. Chlorinated pesticides are widespread chemicals in the former century’s environment. They were used intensively to control agricultural and public health insects in all over the world and still in use in some countries to control malaria disease insect vector. These chemicals might affect newborns and infants when exposed to these pollutants through placenta and breast feeding causing lower birth weight (Siddiqui et al., 2003), neuro-developmental delay (Ribas-Fitó et al. 2003), and disturbance of thyroid hormone (Nagayama et al., 1998). However, these POPs have been banned in Jordan for agricultural insect control in early eighties and for public health purposes in mid-nineties of the last century (Al-Antary, 1996). Pesticides such as DDT, HCH and other organochlorines were also used to control ectoparasites on animals in Jordan and other world countries during the last century (Al-Antary, 1996; Ntow et al., 2006). The hazardous effect of these POPs on environment, wildlife and human beings led to their abuse in Europe, USA and other countries including Jordan in 1970s, 1980s and 1990s (Al-Antary, 1996; Carson, 2002; Clarke et al., 1997).

The ministry of environment in Jordan has asked several authorities for monitoring pesticides residues in the main components of the Jordanian environment. They financed the Royal Scientific Society to monitor chlorinated pesticides residues in human milk in southern Jordan in 2012/2013.

The aim of this study is to monitor the status of these organochlorine pollutants and their main analogous in human breast milk sampled in five districts distributed in southern part of Jordan in 2012/2013.

Materials and methods

Sampling and sample handling

One hundred human breast milk samples were collected in 2012/2013 from five southern districts in Jordan as follows: 25 samples from Karak, 20 samples from Tafilah, 20 samples from Ma’an, 20 samples from Aqaba and 15 samples from Ghor El-Safi. The protocol of this research project has been approved by the participants themselves and a constituted Ethics Committee of the institutions within which the work has been conducted.

An amount of 20–30 mL of breast milk were collected from each donor mother and poured in a 50-mL clean and dry glass bottle. These samples were transferred in a cold ice box to the lab and then stored at −20 °C until analysis. Data concerning age, weight, job, smoking habits, number of deliveries, last delivery date, fat food intake, exposure to pesticides and sampling date for each woman were recorded in a prepared questionnaire.

The mother’s milk samples were gathered in cooperation with the ministry of health employees. The number of samples, which were collected from the cities, towns and villages in the five districts, was determined according to the population densities. However, information on the demographic characteristics of the mothers has been collected particularly; location, date of collection, age, weight, job, smoking, fat recipes, number of birth, last delivery, pesticides exposure and the total detected pesticides residues in mg/kg fat.

Chemicals, solvents and gases

Acetone, dichloromethane and petroleum ether (40–60 °C) were all of p.a. quality whereas n-hexane used was of GC-quality. Standards of the individual chlorinated pesticides were of purity between 99.5% and 99.9%. Anhydrous sodium sulfate was heated at 550 °C for 2 h. Florisil (p.a., 60–100 mesh) was heated at 550 °C overnight, mixed with distilled water to give 3% (w/w), mixed well and kept for 12 h in a closed container prior to use. Helium (99.99% purity) and make-up gas argon/methane (95 + 5%; 99.9% purity) were used for GC analyses. Elution mixture was petroleum ether + dichloromethane (80:20, v/v).

Methods and GC-analysis

Extraction, clean-up and determination were carried out according to DFG-method (Deutsche Forschungsgemeinschaft, 1987), with the following details. Glassware was dried at 110 °C after washing with water, soap, distilled water, acetone and n-hexane. The gas chromatograph (HP-5890) was equipped with 63Ni-electrcapture detector, a splitter injector, and two columns: column I (moderately polar): HP-608 capillary column (30 m, I.D. = 0.25 mm, film thickness = 0.25 μm) and column II (non-polar): HP-5 capillary column (30 m, I.D. = 0.25 mm, film thickness = 0.25 μm). The GC was used under the following operating conditions: injector (280 °C), detector (300 °C), column temperature program: 80 °C (2.2 min), 80–175 °C (30 °C/min), 175–225 °C (10 °C/min) and 225 °C (2 min). Carrier gas (He): 2 mL/min, make-up gas: 30 mL/min, split-ratio: (1:25). These conditions were valid for both used columns.

Determination of fat content

The percent of fat content was determined according to several authors (Deutsche Forschungsgemeinschaft, 1987; MAFF and HSE, 1992). Ten grams of each milk sample were weighed and mixed thoroughly in a separator funnel with 2 mL of 25% ammonia, and then 25 mL diethyl ether with 25 mL petroleum ether (40–60 °C) were added and mixed thoroughly. The organic solvents layer was separated and the previous extraction steps were repeated twice. The pooled organic extracts were filtered through an anhydrous sodium sulfate layer into a weighed round bottom flask. The solvents were evaporated using a rotary evaporator at 30 °C and 200 mbar. The round bottom flask with the residues was placed overnight in a desiccator. Then, the round bottom flask with residues was reweighed, and from the weight difference, the percent of fat content in milk was calculated.

Residues extraction

Twenty five grams Florisil were added to chromatography column (50 × 2 cm, with Teflon stopcock) containing 100 mL petroleum ether. Ten grams milk sample mixed with 25 g Florisil (3% water) were added to the column and the excess solvent was collected in a 500-mL round bottom flask. The column was eluted with 300 mL of the elution mixture. The eluate was rotary-evaporated nearly to dryness at 35 °C and 12 mbar. The remaining solvent was evaporated using a stream of nitrogen gas. The residues were dissolved in 2 mL n-hexane containing 0.3 μg/mL of the internal standard (I.S) aldrin, and 2 μL of the final extract were injected onto the GC column.

Recovery tests and detection limits

Extraction and clean-up method was evaluated by spiking blank milk samples with known concentrations of each of the studied chlorinated pesticides, and each of these samples was extracted and cleaned-up according to the above-mentioned method. The experimentally found concentration was related to the theoretically added concentration to calculate the percent of recovery. The detection limit for each compound was calculated as S/N ≥ 3 from the chromatogram of the standard mixture of the 16 studied pesticides after dilution for several times. Each solution was injected twice. The results of percent of recovery and detection limits are shown in Table 1.

Table 1. Mean recovery (%) and mean detection limits for the studied chlorinated pesticides in mother milk samples from southern Jordan in 2012/2013.

Pesticide	Mean detection limit (μg/mL)	% Recovery
Aldrin	0.005	99.0
Dieldrin	0.005	87.8
Endrin	0.005	79.9
α-endosulfan	0.005	94.5
β-endosulfan	0.005	99.2
HCB	0.005	79.3
α-HCH	0.005	94.8
β-HCH	0.005	91.9
γ-HCH	0.005	94.1
Heptachlor	0.005	99.0
o,p′-DDE	0.005	94.8
o,p′-DDD	0.005	86.9
o,p′-DDT	0.005	94.7
p,p′-DDD	0.005	90.3
p,p′-DDE	0.005	98.7
p,p′-DDT	0.005	90.3

Results

Results in Table 2 show the amount and range (minimum and maximum) of pesticides residues for all samples in Karak district in 2012/2013, in addition to the number and percentage of contaminated samples for each chlorinated pesticide. Only three compounds were detected in the samples, these were p,p’-DDE (56% and average of 0.21 mg/kg milk fat), β-HCH (36% and average of 0.13 mg/kg) and p,p’-DDT (16% and average of 0.04 mg/kg).

Table 2. Average and range of chlorinated pesticides residues in mother milk samples from Karak district in Jordan in 2012/2013.

Chlorinated pesticide	N	N%	Average concentration (mg/kg fat)	Concentration range (mg/kg fat)
Aldrin	–	–	–	–
Dieldrin	–	–	–	–
Endrin	–	–	–	–
α-endosulfan	–	–	–	–
β-endosulfan	–	–	–	–
HCB	–	–	–	–
α-HCH	–	–	–	–
β-HCH	9	36	0.13	0.06–0.83
γ-HCH	–	–	–	–
Heptachlor	–	–	–	–
o,p′-DDE	–	–	–	–
o,p′-DDD	–	–	–	–
o,p′-DDT	–	–	–	–
p,p′-DDD	–	–	–	–
p,p′-DDE	14	56	0.21	0.04–1.43
p,p′-DDT	4	16	0.04	0.08–0.35

Notes: N = 25, average fat content = 3.5%, N = number of positive samples, N% = percentage of positive samples.

Results in Table 3 show the amounts and range (minimum and maximum) of pesticides residues for all samples in Tafilah district in 2012/2013, in addition to, number and percentages of contaminated samples for each chlorinated pesticide. Four compounds were detected in the samples. These chemicals were p,p’-DDE (30% and average of 0.07 mg/kg milk fat), β-HCH (25% and average of 0.1 mg/kg milk fat), α-HCH (10% average of 0.008 mg/kg milk fat) and heptachlor (5% and average of 0.004 mg/kg milk fat).

Table 3. Average and range of chlorinated pesticides residues in mother milk samples from Tafilah district in Jordan in 2012/2013.

Chlorinated pesticide	N	N%	Average concentration (mg/kg fat)	Concentration range (mg/kg fat)
Aldrin	–	–	–	–
Dieldrin	–	–	–	–
Endrin	–	–	–	–
α-endosulfan	–	–	–	–
β-endosulfan	–	–	–	–
HCB	–	–	–	–
α-HCH	2	10	0.008	0.08
β-HCH	5	25	0.1	0.16–0.64
γ-HCH	–	–	–	–
Heptachlor	1	5	0.004	0.08
o,p′-DDE	–	–	–	–
o,p′-DDD	–	–	–	–
o,p′-DDT	–	–	–	–
p,p′-DDD	–	–	–	–
p,p′-DDE	6	30	0.07	0.1–0.77
p,p′-DDT	–	–	–	–

Notes: N = 20, average fat content = 3.0%, N = number of positive samples, N% = percentage of positive samples.

Results in Table 4 show the amounts and range (minimum and maximum) of pesticides residues for all samples in Ma’an district in 2012/2013, in addition to, number and percentages of contaminated samples for each chlorinated pesticide. Five compounds were detected in the samples. These chemicals were p,p’-DDE (55% and average of 0.14 mg/kg milk fat), p,p’-DDT (15% and average of 0.01 mg/kg milk fat), β-HCH (10% and average of 0.04 mg/kg milk fat) and heptachlor (5% and average of 0.02 mg/kg milk fat).

Table 4. Average and range of chlorinated pesticides residues in mother milk samples from Ma’an district in Jordan in 2012/2013.

Chlorinated pesticide	N	N%	Average concentration (mg/kg fat)	Concentration range (mg/kg fat)
Aldrin	–	–	–	–
Dieldrin	–	–	–	–
Endrin	1	5	0.02	0.56
α-endosulfan	–	–	–	–
β-endosulfan	–	–	–	–
HCB	–	–	–	–
α-HCH	–	–	–	–
β-HCH	2	10	0.04	0.23–0.72
γ-HCH	–	–	–	–
Heptachlor	1	5	0.007	0.14
o,p′-DDE	–	–	–	–
o,p′-DDD	–	–	–	–
o,p′-DDT	–	–	–	–
p,p′-DDD	–	–	–	–
p,p′-DDE	11	55	0.14	0.07–0.97
p,p′-DDT	3	15	0.01	0.08–0.11

Notes: N = 15, average fat content = 3.2%, N = number of positive samples, N% = percentage of positive samples.

Results in Table 5 show the amounts and range (minimum and maximum) of pesticides residues for all samples in Aqaba district in 2012/2013, in addition to the number and percentages of contaminated samples for each chlorinated pesticide. Two compounds were detected in the samples. These chemicals were p,p’-DDE (65% and average of 0.18 mg/kg milk fat) and β-HCH (30% and 0.13 mg/kg milk fat).

Table 5. Average and range of chlorinated pesticides residues in mother milk samples from Aqaba district in Jordan in 2012/2013.

Chlorinated pesticide	N	N%	Average concentration (mg/kg fat)	Concentration range (mg/kg fat)
Aldrin	–	–	–	–
Dieldrin	–	–	–	–
Endrin	–	–	–
α-endosulfan	–	–	–	–
β-endosulfan	–	–	–	–
HCB	–	–	–	–
α-HCH	–	–	–	–
β-HCH	6	30	0.13	0.03–0.59
γ-HCH	–	–	–	–
Heptachlor	–	–
o,p′-DDE	–	–	–	–
o,p′-DDD	–	–	–	–
o,p′-DDT	–	–	–	–
p,p’-DDD	–	–	–	–
p,p′-DDE	13	75	0.18	0.08–0.77
p,p′-DDT	–	–		–

Notes: N = 20, average fat content =2.9%, N = number of positive samples, N% = percentage of positive samples.

Results in Table 6 show the amounts and range (minimum and maximum) of pesticides residues for all samples in Ghor Al-Safi district in 2012/2013, in addition to the number and percentages of contaminated samples for each chlorinated pesticide. Four compounds were detected in the samples. These chemicals were p,p′-DDE (100% and average of 0.73 mg/kg milk fat), β-HCH (53% and 0.21 mg/kg milk fat), α-HCH (27% and average of 0.04 mg/kg) and p,p′-DDT (20% and average of 0.09 mg/kg).

Table 6. Average and range of chlorinated pesticides residues in mother milk samples from Ghor Al-Safi district in Jordan in 2012/2013.

Chlorinated pesticide	N	N%	Average concentration (mg/kg fat)	Concentration range (mg/kg fat)
Aldrin	–	–	–	–
Dieldrin	–	–	–	–
Endrin	–	–	–
α-endosulfan	–	–	–	–
β-endosulfan	–	–	–	–
HCB	–	–	–	–
α-HCH	4	27	0.04	0.11–0.15
β-HCH	8	53	0.21	0.13–1.68
γ-HCH	–	–	–	–
Heptachlor	–	–
o,p′-DDE	–	–	–	–
o,p′-DDD	–	–	–	–
o,p′-DDT	–	–	–	–
p,p′-DDD	–	–	–	–
p,p′-DDE	15	100	0.73	0.10–1.50
p,p′-DDT	3	20	0.09	0.10–0.88

Notes: N = 15, average fat content =3.2%, N = number of positive samples, N% = percentage of positive samples.

Results in Table 7 show the amounts and range (minimum and maximum) of pesticides residues for all the 100 samples in the five districts in 2012/2013, in addition to, number and percentages of contaminated samples for each chlorinated pesticide. Six compounds were detected in the whole 100 samples. These chemicals were p,p′-DDE (59% and average of 0.24 mg/kg milk fat), β-HCH (30% and 0.12 mg/kg milk fat), α-HCH (6% and average of 0.007 mg/kg), p,p’-DDT (10% and average of 0.02 mg/kg), heptachlor (2% and 0.002 mg/kg) and endrin (1% and 0.005 mg/kg).

Table 7. Average and range of chlorinated pesticides residues in all studied mother’s milk samples collected from five districts in Jordan in 2012/2013.

Chlorinated pesticide	N	N%	Average concentration (mg/kg fat)	Concentration range (mg/kg fat)
Aldrin	–	–	–	–
Dieldrin	–	–	–	–
Endrin	1	1	0.005	0.56
α-endosulfan	–	–	–	–
β-endosulfan	–	–	–	–
HCB	–	–	–	–
α-HCH	6	0.007	0.08	0.15
β-HCH	30	30	0.12	0.005–1.68
γ-HCH	–	–	–	–
Heptachlor	2	2	0.002	0.08–0.14
o,p′-DDE	–	–	–	–
o,p′-DDD	–	–	–	–
o,p′-DDT	–	–	–	–
p,p′-DDD	–	–	–	–
p,p′-DDE	59	59	0.24	0.04–1.50
p,p′-DDT	10	10	0.002	0.08–0.88

Notes: N = 15, average fat content =3.2%, N = number of positive samples, N% = percentage of positive samples.

Discussion

Breast milk is important for newborns and infants well physical and mental developing. Unfortunately, unintentional pollutants such as chlorinated pesticides in our environment through food chain, air and water as a result of eating and drinking might reach milk. The mentioned tables in the results show that 1% of the samples which contained pesticides residues was endrin, 6% contained α-HCH, 30% contained β-HCH, 2% contained heptachlor, 59% contained p,p’-DDE and 10% contained p,p’-DDT. Zeinab et al. (2011) mentioned that most mother-milk samples in El-Gabal Al-Akhdar in Libya contain DDT, BHC, endrin and heptachlor. Stuetz et al. (2001) found that all mother-milk samples contained DDT in northern Thailand.

The tables show that the maximum residues were for p,p′-DDE (1.50 mg/kg milk fat) in Ghor Al-Safi district. The maximum average for pesticide residues in all samples was 0.72 mg/kg milk fat in Ghor Al-Safi. The maximum percentage for pesticides residues was 10% which was also in Ghor Al-Safi. Alawi et al. (2013) reported that the highest average residues found was for p,p′-DDT followed by β-HCH in Karak and Ghor Al-Safi in southern part of Jodan.

The present study showed that all collected samples from the five districts in southern Jordan in 2012/2013 were free of aldrin, dieldrin, α-endosulfan, β-endosulfan, HCB, γ-HCH, o,p′-DDD, o,p′’-DDT and o,p′-DDE. Al-Antary et al. (2013) in a study conducted in 2005/2006 in south districts of Jordan found no detected residues for heptachlor, o,p′-DDE, p,p′-DDD, endrin, aldrin and dieldrin in the collected mother-milk samples and no detectable residues for γ-HCH, heptachlor, o,p′-DDD, p,p′-DDD, o,p′-DDE, o,p′-DDT, p,p′-DDT, endrin, aldrin and dieldrin in the same part of Jordan in 2007/2008.

In Ghana, Tutu et al. (2011) found that all collected mother-milk samples were contaminated with all pollutants mentioned in the present study. In Hawaii, Takahashi et al. (1981) reported that all analyzed human milk samples contained p,p′-DDT, heptachlor epoxide, dieldrin and HCH. In Egypt, Dogheim et al. (1996) showed that aldrin, dieldrin, heptachlor, heptachlor epoxide and endrin residues have been eliminated from human milk in Kafr El-Zayal governate. However, the results of this study is approximately similar to the results studies conducted in Sweden concerning DDTs in breast milk (MAFF and HSE, 1992) and the residues levels of dieldrin, heptachlorepoxide and DDTs in the United States (Calabrese, 1982). In addition, the results of the present study are similar to those obtained from Jordan by several workers (Alawi et al., 2013) on breast milk from mothers living in Amman, the capital of Jordan.

In conclusion, six chlorinated pesticide have been detected in all mother-milk samples including endrin, α-HCH, β-HCH, heptachlor, p,p′-DDE and p,p′-DDT particularly in Ghor Al-Safi district. This district is intensively cultivated with vegetables such as tomatoes, pepper, eggplant, cucumber and other vegetables from solanacious, cucurbits and cruceters.

The present study recommends the continuous pesticide-residues monitoring in all parts of Jordan particularly Ghor Al-Safi and other regions in cultivated Jordan valley.

Acknowledgements

The authors greatly acknowledge the University of Jordan and Ministry of Environment for help and the Royal Scientific Society for analyzing the milk samples.

Declaration of interest

The authors declare that they have no conflict of interest. This study was funded by Ministry of Environment.

Compliance with Ethical Standards

Informed consent was obtained from all individual participants included in the study.

The protocol of this research project has been approved by a constituted Ethics Committee of the institutions within which the work has been conducted.