Environmental Exposures during Lactation: Mycotoxins and Pesticides in Human Milk from Haryana, India Open Access

Abstract

Exclusive breastfeeding in the first six months of life is recommended globally as breastmilk contains nutrients and immune factors essential for child growth and development. Breastmilk can also carry contaminants such as mycotoxins and pesticides. We aimed to examine the prevalence of these exposures in breastmilk and food samples from Haryana, India.

We developed and validated a method to quantify 6 aflatoxins (B1, B2, G1, G2, M1, M2) and 2 Ochratoxins (A & B) and tested it on four different types of animal milk samples (n = 38) collected from southern India, using ultra-high-performance liquid chromatography tandem mass spectrometry. Method performance parameters were in accordance with USFDA guidelines and LOQs ranged between 15.6 and 156 pg/mL. Using this method, we quantified AFB2 in goat milk (mean: 38 pg/mL), AFM1 in cow (331 pg/mL), goat (406 pg/mL), and pasteurized milk (164 pg/mL), and 90% of cow, goat and pasteurized milk samples were above EU limits of 50 pg/mL.

           Next, we quantified 8 co-occurring mycotoxins in human breastmilk (infants 2-4 months) samples (n = 100) using UHPLC-MS/MS and ELISA to detect aflatoxin B1, fumonisin B1, and deoxynivalenol in several food commodities (n = 298). To the best of our knowledge, we present the first study to quantify mycotoxins in breastmilk from India. We found low concentrations of AFM1 in 41% (> LOD) of samples (median: 13.7; range: 3.9-1200 pg/mL. AFM1 was detected above FSSAI regulatory limits (500 pg/mL) in 27% of animal milk samples (n = 30). Maternal consumption of breads was associated (β = 0.32; 95% CI: 0.22, 10.99, p < 0.05) with breastmilk AFM1 exposure. AFB1 was detected in several food commodities including wheat (median: 1.9 µg/kg; 0-196), rice (0 µg/kg; 0-195.6), flour (3.13 µg/kg; 0-214.9). FB1 was detected in maize, pearl millet and sorghum. DON was not detected in our study. Estimated daily intakes for 80% and 100% of women exceeded PMTDI cut-offs for AFM1 consumption via animal milk and AFB1 consumption in rice and flour (mean: 75.81; range: 35.2-318.2 ng/kg bw/day), respectively. Calculated daily intakes for AFM1 in breastmilk exceeded PMTDI (1 ng/kg bw/day) recommendations for 40% of infants (mean 1.63; range: 0.26-18.20 ng/kg bw/day).

The third objective was to quantify 53 organochlorine, organophosphate and pyrethroid pesticides in breastmilk samples (n = 75) using GC-MS and GC-ECD. We used 30 in-depth interviews and 9 focus group discussions to assess maternal and community perceptions, knowledge, attitudes and practices associated with pesticide use. We detected p,p’-DDT and p,p’-DDE in 4% (range: < LOQ-28 µg/L) and 5% (range: < LOQ-107 µg/L) of samples, respectively. Findings suggest gendered engagement in farming, which precludes pesticide and fertilizer handling and use by lactating women. Chemicals are widely used despite varying perceptions of them as both medicines and poison, recognizing their need to ensure food security.

Although we did not detect pesticide concentrations of significant public health risk, we did quantify mycotoxins in human milk and food samples. Our findings suggest a need for further investigation of exposures across the food chain and associated health impacts.  

Table of Contents

Chapter 1: Introduction……………………………………………………….…………………1

1.1 Specific Aims. ………………………………………………………………………………...4

Chapter 2: Literature review.. …………………………………………….……………………8

2.1 Child growth. ………………………………………………………………………………….9

2.1.1 Exclusive breastfeeding and breastmilk composition. ……………………………14

Exclusive Breastfeeding. …………………………………………………….………………….14

Breastmilk Composition. ……………………………………………………………………….15

2.1.2 Environmental Enteric Dysfunction. …………………………………..……………..17

Biomarkers of EED.. ……………………………………………………………..……………….19

2.2 Mycotoxins. ……………………………………………………………………..……………..21

2.2.1 Food systems, prevention and mitigation. …………………………………………...23

1) Aflatoxins. …………………………………………………………………..…………………..23

2) Ochratoxins. ……………………………………………………………………………………..24

3) Fumonisins. ……………………………………………………………………………………...25

4) Deoxynivalenol …………………………………………………………….……………………26

2.2.2 Interactions between multiple mycotoxins. ……………….………………………….26

2.2.3 Regulatory limits. ………………………………………………..………………………….28

2.2.4 Hazard identification & characterization and Exposure assessment …………...32

Hazard Assessment ………………………………………………………………………………..32

Risk Characterization. …………………………………………………………………………….33

2.2.5 Health outcomes. …………………………………………………………………………...36

2.2.6 Association with pregnancy outcomes. …………………………………………..…...39

Maternal Outcomes. ……………………………………………………………………………….39

Child Outcomes. ……………………………………………………………………………………40

2.2.7 Breastmilk and infant foods.........................................................................41

2.2.8 Associations with child growth outcomes. ....................................................44

2.3 Pesticides......................................................................................................49

2.3.1 Food systems, prevention and mitigation......................................................51

1) Organochlorines..............................................................................................51

2) Organophosphates...........................................................................................54

3) Pyrethroids..................................................................................................... 55

2.3.2 Regulatory limits......................................................................................... 56

2.3.3 Hazard identification & characterization and Exposure assessment ................61

2.3.4 Health outcomes......................................................................................... 64

2.3.5 Association with pregnancy outcomes.......................................................... 66

Maternal Outcomes............................................................................................ 66

Child Outcomes................................................................................................. 68

2.3.6 Breastmilk and infant foods........................................................................ 71

2.3.7 Associations with child growth outcomes.................................................... 74

2.4 Methods for Assessment of Mycotoxins & Pesticides....................................... 77

Mycotoxins........................................................................................................ 78

2.4.1a Biomarkers and matrices........................................................................... 78

2.4.2a Sample preparation and analytical methods............................................... 80

2.4.3a Comparisons of LODs, LOQs and method performance................................ 83

Pesticides........................................................................................................... 87

2.4.1b Biomarkers and matrices............................................................................87

2.4.2b Sample preparation and analytical methods................................................ 88

2.4.3b Comparisons of LODs, LOQs and method performance................................. 90

2.5 Summary and research gaps............................................................................ 97

Chapter 3: Quantification of aflatoxin and ochratoxin contamination in animal milk using UPHPLC-MS/SRM method: a small-scale study.........98

Abstract............................................................................................................101

1. Introduction................................................................................................. 102

2. Experimental ................................................................................................104

3. UHPLC-MS.................................................................................................... 105

4. Results.......................................................................................................... 109

5. Discussion..................................................................................................... 117

6. Conclusion.................................................................................................... 120

7. References......................................................................................................122

Chapter 4: Risk of dietary and breastmilk exposure to mycotoxins among lactating women and infants 2-4 months in Northern India....................................128

Abstract .............................................................................................................129

1. Introduction....................................................................................................131

2. Materials and Methods.................................................................................... 133

3. Results........................................................................................................... 142

4. Discussion.......................................................................................................151

5. Conclusion.......................................................................................................157

6. References........................................................................................................158

Chapter 5: A Mixed-Methods Study of Pesticide Exposures in Breastmilk and Community & Lactating Women’s Perspectives from Haryana, India....................................174

Abstract ………………………………………………………………………………………...175

1. Background.............................................................................................. 178

2. Material and Methods................................................................................179

3. Results.....................................................................................................188

4. Discussion................................................................................................197

5. Conclusions..............................................................................................200

6. References............................................................................................... 202

Chapter 6: Mediation path analyses of associations between breastmilk mycotoxins and infant growth outcomes......................................211

Abstract.......................................................................................................212

1. Introduction............................................................................................. 213

2. Materials & Methods..................................................................................216

3. Results......................................................................................................220

4. Discussion................................................................................................ 227

5. References.................................................................................................232

Chapter 7: Discussion....................................................................................237

7.1 Summary of Key Findings......................................................................... 237

7.2 Strengths & Limitations.......................................................................... 243

Strengths.................................................................................................... 243

7.2.1 Study design, sample size and sample collection..................................... 243

7.2.2 Methods and data breadth.....................................................................245

7.2.3 Analytical and statistical methods......................................................... 247

Limitations..................................................................................................249

7.2.4 Selection bias and generalizability.........................................................249

7.2.5 Study design and biomarkers.................................................................251

7.2.6 Sample size and power..........................................................................253

7.2.7 Analytical limitations............................................................................254

7.3 Future Studies & Next Steps.....................................................................255

7.3.1 Leveraging existing study samples..........................................................256

7.3.2 Longitudinal cohort study.....................................................................260

7.3.3 Randomized controlled trial/intervention study. ....................................263

Chapter 8: Conclusion..................................................................................268

Chapter 2, 7, 8 References.............................................................................270

About this Dissertation

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School	Laney Graduate School
Department	Nutrition and Health Sciences
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Health Sciences, Nutrition
Keyword	Breastmilk Pesticides Mycotoxins
Committee Chair / Thesis Advisor	Melissa Fox Young, Emory University
Committee Members	P. Barry Ryan , Emory University Usha Ramakrishnan, Emory University Amy Webb Girard, Emory University Reynaldo Martorell, Emory University

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