Tiny pesticide exposure during pregnancy can have long-term impact on female offspring

Study confirms chlorpyrifos levels far below "toxic" threshold can impair learning, change brain function and alter thyroid levels into adulthood for tested mice.

By Greg Bowman
A new animal study accentuates the risk of ultra-low levels of the common pesticide chlorpyrifos to cause long-lasting birth defects in female offspring of exposed mothers. The daughters exhibited learning delays, disturbed brain function and altered thyroid levels. Significantly, these symptoms resulted from low toxicity exposure during late gestation—an impact route not even part of current regulatory pesticide testing.

Damage at these doses highlights vulnerability during gestation from toxins even at the parts per billion level.

In a paper published October 29 by Reproductive Toxicology (1), a peer-reviewed academic journal, a team of researchers from the University of Wisconsin, Department of Zoology, detail their findings from research on pregnant female mice exposed to minute levels of chlorpyrifos late in gestation.

Corresponding researcher is Warren Porter, Ph.D., professor of zoology and environmental toxicology. He’s a former departmental chair, and has studied in many others fields. He was the first faculty associate at the university’s Center for Integrated Agricultural Systems with a home department outside of College of Agriculture and Life Sciences.

The results are dramatic because of the tiny doses involved, and because of the near ubiquity of chlorpyrifos present in humans. The pesticide is highly regulated because of its known hazards to human health, but is still used widely in food production, ornamental horticulture and treating buildings for insects.

Chlorpyrifos (CPF) is used in liquid termite soil treatments; as lawn/garden granules, dusts, and sprays; in household aerosols, baits, dusts, and sprays; in agricultural sprays and granules (Durban and Lorsban); and in animal flea collars, dips, and sprays. See the U.S. Environmental Protection Agency’s rap sheet on the chemical here.

Risk from pesticides is already tested in many ways, but generally for acute toxicity (poisoning) or the risk to cause cancer, reproductive, neurological, metabolic or birth defects to due to a high dose. The Wisconsin study follows on a growing body of work showing disturbing impacts at ultra-low levels that seem to mimic hormones, and therefore slip through the body’s protective systems that are designed to counteract toxins.

The EPA sheet above states: “Dietary exposures from eating food crops treated with chlorpyrifos are below the level of concern for the entire U.S. population, including infants and children.” Porter and his associates show that is likely not the case, if the usual level of relevance of mouse response to predictable human response holds true in this case. The EPA recently announced further endocrine testing protocol for pesticides and other materials.

The research
Pregnant mice were injected with 0, 1 or 5 mg CPF per kg of body weight. Their offspring were evaluated for several types of learning ability in a foraging maze from the age of 60 days to 150 days of age. The mice were evaluated for their ability to find food, how fast they found it, how well they remembered where it was. Thyroid hormone levels were checked at the end of the test.

Results demonstrated “a long-term, dose dependent, sex selective impairment of foraging behavior and as well as learning latency in female mice exposed to CPF in utero.” The traces of pesticide, even at the lower 1 mg CPF/kg of body weight level, did not impact the learning ability male mice, but had significant impacts on the females. Further, the CPF dosing of their mothers did not change the serum thyroid hormone level of the male mice, but correlated directly to the mother’s dose in female offspring. The detrimental changes persisted into adulthood for the female mice.

Porter points out that most pesticide testing is done on male rats, which are probably the most resistant to showing response to toxins, while female mice may be the most sensitive.

Females exhibited “diminished foraging ability in a dose dependant manner due to in utero CPF exposure,” the paper says. In food recognition and food position learning assessments (a novel food in a novel place), the young mice with no exposure learned at a steady rate, while the lower-dosed group took longer to reach the same level. The higher dose group never did attain the same level of success in these foraging ability tests.

Chlorpyrifos background
The University of Wisonsin authors provided this background from other research in their paper’s discussion section:

• Chlorpyrifos may represent a significant source of in home pesticide exposure via direct contact and non-organic food consumption (2) 
• Authors in a broad literature review of chlorpyrifos and other organophosphorus pesticides show a long history of impacts on DNA synthesis in the brain, altered neurological functions, and impacts beyond the nervous system.(3)
• Even though organophosphates are thought to be cleared quickly after exposure of ingestion, the consistent presence of chlorpyrifos metabolites in the urine of children on conventionally produced diets suggests frequent repeated exposures via diet. (4) .

After tracing two mechanisms that could explain how in utero chlorpyrifos exposure could influence neuron development (impacting brain function and learning ability), the authors say that their work, combined with other cited findings, suggests that CPF-treated brains may form alternate neural pathways, functioning differently than normal brains.

The writers, in conclusion:

• Say their study “contributes to the growing body of evidence that demonstrates the need for CPF and other organophosphates to be analyzed for endocrine disruption during risk assessment analyses.”
• Hypothesize that altered neural pathways (from CPF exposure) may be located in a brain region that controls both behavior and thyroid hormone levels.
• Write that study reinforces the idea that low dose in utero exposures must be considered during toxicity assessment of materials.

The intergenerational connections shown between a pesticide-exposed mother and an impacted daughter are profound.  The daughter’s neurological alterations, that may impact her learning ability and master hormone—thyroid—are troubling, indeed.

Low-dose dangers
This study accentuates the relatively unregulated universe of low-level impact risk from pesticides and other sources of toxics. The mantra “the dose makes the poison” has diverted rulemaking for toxins to focus on threshold testing of direct impact, i.e. what is minimum amount of an active ingredient that will trigger a measurable impact on the exposed organism or its reproductive capacity. The mode of action is almost the opposite with extremely low, sub-lethal doses. These are present in concentrations at orders of magnitude less than the toxic threshold, and gain their damaging impact by mimicking hormone function.

Some “dose” context helps to understand what may be at stake.

• For chlorpyrifos to function in its direct toxic mode takes a dose somewhere more than 50 parts per billion (ppb). There is no demonstrated cholinesterase inhibition at 5 to 50 ppb for the toxin, according to a study of chlorpyrifos workers published in 2008. (5) 
• An important 2006 study (4) found background levels of about 20 ppb of pesticide biomarker for chlorpyrifos in children’s urine. This dropped to “non-detectable” immediately after the children were switched being fed organic food, then went back up when they resumed their regular eating habits.
• The chlorpyrifos dose in this study, Haviland et. al., were 1 and 5 parts per million (ppm), which is a range 1,000 times higher than that found in the children. Impacts in Haviland et al, were dose-dependent, gender-linked and continued into adulthood, including learning disability and thyrroid level.
• Human estrogen levels fluctuate between 40 and 400 parts per trillion (ppt), which are also representative of the concentration ranges our bodies respond to chemicals.
• There is no required regulatory testing for agricultural pesticide active ingredient impact on human health based on exposure at the ultra-low level.

The Haviland, et al, paper in Reproductive Toxicology will be accessible here.

Full disclosure: Funders listed for this research project are the Rodale Institute, the Lumpkin Foundation and the Bradshaw-Knight Foundation.

Greg Bowman is communications manager for the Rodale Institute

Story citations
1. Haviland JA, et al. Long-term sex selective hormonal and behavior alterations in mice exposed to low doses of chlorpyrifos in utero. Reproductive Toxicology (2009), doi:10.1016/j.reprotox.2009.10.008

2. Eskenazi B, Bradman A, Castorina R. Exposures of children to organophosphate pesticides and their potential adverse health effects. Environ Health Perspect 1999;107(Suppl 3):409–19.

3. Slotkin TA. Developmental cholinotoxicants: nicotine and chlorpyrifos. Environ Health Perspect 1999;107(Suppl 1):71–80.

4. Lu C, et al. Organic diets significantly lower children’s dietary exposure to organophosphorus pesticides. Environ Health Perspect 2006;114(2): 260–3.

5. David H Garabrant, et al. Cholinesterase inhibition in chlorpyrifos workers: Characterization of biomarkers of exposure and response in relation to urinary TCPy. Journal of Exposure Science and Environmental Epidemiology (2009) *19,* 634–642; doi:10.1038/jes.2008.51; published online 20 August 2008.

Relevant research
Resource citations relating to human health impacts of chlorpyrifos, issued November 6, 2009, by the National Pesticide Information Center.

Smegal, D. C. Human Health Risk Assessment Chlorpyrifos;
U.S. Environmental Protection Agency, Office of Prevention,
Pesticides and Toxic Substances, Office of Pesticide Programs, Health
Effects Division, U.S. Government Printing Office: Washington, DC, 2000; pp 1-131

Ricceri, L., Markina, N., Valanzano, A., Fortuna, S., Cometa, M.F.,
Meneguz, A., Calamandrei, G.
Developmental exposure to chlorpyrifos alters reactivity to environmental and social cues in
adolescent mice. Toxicol. Appl. Pharmacol. 2003, 191, 189-201.

Zheng, Q., Olivier, K., Won, W.K., Pope, C.N.
Comparative Cholinergic Neurotoxicity of Oral Chlorpyrifos Exposures in Preweanling and Adult Rats. Toxicol. Sci. 2000, 55, 123-132.

Moser, V. C.; Padilla, S.
Age- and Gender-Related Differences in the Time Course of Behavioral and Biochemical Effects
Produced by Oral Chlorpyrifos in Rats. Toxicol. Appl. Pharmacol. 1998, 149, 107-119.

Dam, K., Seidler, F.J., and Slotkin, T.A.
Chlorpyrifos exposure during a critical neonatal period elicits gender-selective
deficits in the development of coordination skills and locomotor activity.
Dev. Brain Res. 2000, 121 (2), 179-187.

Carr, R. T.; Chambers, H.W.; Guarisco, J. A.; Richardson, J. R.; Tang,
J.; Chambers, J. E. 
Effects of Repeated Oral Postnatal Exposure to Chlorpyrifos on Open-Field Behavior in Juvenile Rats. Toxicol. Sci. 2001, 59, 260-267.

Jett, D. A.; Navoa, R. V.; Beckles, R. A.; McLemore, G. L. Cognitive
Function and Cholinergic Neurochemistry in Weanling Rats Exposed to Chlorpyrifos. Toxicol. Appl. Pharmacol. 2001, 174 (2), 89-98.

Roy, T. S.; Andrews, J. E.; Seidler, F. J.; Slotkin, T. A.
Chloropyrifos Elicits Mitotic Abnormalities and Apoptosis in
Neuroepithelium of Cultured Rat Embryos. Teratology 1998, 58, 62-68.