Greenlee , AR , TM Ellis, RL Berg. 2004. Low-Dose Agrochemicals and Lawn-Care Pesticides Induce Developmental Toxicity in Murine Preimplantation Embryos. Environmental Health Perspectives 112: 703-709.

Exposure to very low doses of common pesticides, at levels currently assumed to be safe--and within dose ranges that have been measured in people--caused significant injury to mice embryos tested in vitro during early stages of development. Twelve of 13 pesticides tested caused developmental injury while 6 of 6 environmentally-realistic mixtures caused damage.

Why was this study done?

Current protocols for testing chemicals for developmental and reproductive effects have several drawbacks. A major concern is that they may fail to detect some effects resulting from real-world exposures to chemicals. For example, they tend to test chemicals one at a time, at fairly high doses, and do not evaluate risks caused by exposure at a very early stage in development, before an embryo implants in the uterus. Another drawback is the cost and time required to complete the studies. Recent studies indicate the importance of the timing of exposure and exposure at low levels, and to mixtures of chemicals.

This study focused on pre-implantation mouse embryos exposed to chemicals, both individually and in mixtures chosen to simulate real-world exposures. The purpose of such a method was to determine whether or not such exposures would have an effect on development at this early stage. A goal of the study was to determine whether this method was a reliable, rapid, and cost-effective way to evaluate the effects of pesticides at low doses and in combinations potentially encountered before a pregnancy is recognized.

What did they do?

The research team gave female mice hormone injections to make sure there would be available eggs to fertilize. They then housed the female mice with male mice of proven mating ability. After fertilization, the single-celled zygotes were collected before having a chance to implant in the uterine lining of the female mouse.

These one-celled embryos were incubated in vitro for four days while being exposed to either low-dose concentrations of agrochemicals and lawn pesticides, a positive control, or one of two negative controls.

Six herbicides, three insecticides, two fungicides, one desiccant, one fertilizer and six mixtures were tested:

The concentration of chemicals in solution was calibrated to represent the “safe” level (“reference dose”) established by the U.S. Environmental Protection Agency (EPA). The negative controls were used to determine any possible effects that other substances in the solution besides the pesticides might have on embryo development.

After incubation, the scientists examined whether the embryos developed normally. They measured three things: cell apoptosis; the percentage of embryos who developed past the zygote stage to the blastocyst; and the average number of cells making up the embryo. Each of these measures would reflect impacts decreasing the likelihood that an embryo would fully complete development.

What did they find?

As expected from previous studies, the embryos in the positive control group--exposed to DDT--showed apoptosis and reduced development to the blastocyst stage of development.

11 of 13 chemicals tested individually caused a significant increases in apoptosis. The largest percentage increases in apoptosis were caused by the positive control (DDT) or treatment by diquat, mancozeb, atrazine or 2,4-D (graph to right).     3 asterisks: p<0.0005 2 asterisks: p<0.005 1 asterisk: p<0.05

Three of the chemicals--atrazine, chlorpyrifos and terbufos--reduced the number of embryos developing to to blastocysts (all p < 0.05)

Ammonium nitrate reduced the average cell number per embryo (p < 0.0005).

Mecoprop reduced embryo cell number (p < 0.05).

The mixtures tested had varied results. Some caused increased apoptosis (graph to left) Others reduced embryo development and/or lower average cell number in each embryo. While some mixtures showed a pattern of injury similar to the pesticides tested individually, other mixtures showed a different pattern. (see table above for components of mixtures)

• The mixture created to represent preemergent and postemergent herbicides showed effects similar to exposures caused by the individual components of the mixtures: increasing apoptosis with no apparent effects on blastocyst development or embryo cell number.
• Mixtures created to resemble groundwater contamination and lawn care herbicides reduced development to blastocyst and increased apoptosis.
• Both the mixture formulated to represent lawn-care herbicides and the fungicide-dessicant mixture reduced the percentage of embryos which reached the blastocyst stage of development, even though none of the individual chemicals in these mixtures did so.
• While the mixture formulated to represent groundwater contaminants also reduced the percentage of embryos that reached the blastocyst stage, it did not increase apoptosis or influence the mean cell number per embryo, even though some chemicals individually in the mixture did so.

Why is this important?

Greenlee et al. show that embryonic injury can be caused by pesticide exposure doses considered safe by the U.S. EPA, and at or near current human exposure levels, based on other studies measuring pesticides in pregnant women. Their work also demonstrated that damage can occur at extremely early periods of embryonic development, even before the embryo has implanted in the uterine lining. Were these same effects to occur in humans, in all likelihood they would occur before a woman was aware that she was pregnant.

While these experiments were conducted with isolated mouse embryos in vitro, the results are consistent with several studies of the effect of agricultural chemicals on developing mice. For example, Cavieres et al. reported that a common mixture of lawn herbicides (2,4-D, MCPP and dicamba) reduced implantation sites and live births even at very low doses.

Are these results relevant to human health, and in particular, to reproductive health?

• The types of damage examined in this study would adversely affect embryo health and decrease the likelihood of successful birth.
• Exposures to the chemicals used in the experiments are common in people, as they are among the most abundantly used agrochemicals in the upper midwestern US. According to Greenlee et al., the exposure levels they used are within ranges seen in human reproductive tissues and fluids.
• The developmental events underway in humans as the zygote is converting to a blastocyste and then implanting are highly similar to those underway in mice embryos at the same stage of development.
• While difficult to study, research indicates that many human pregnancies are lost at this stage of development. One study reports rates from 21 to 70%. The highest rate was for women with prior indications of subfertility.

These considerations suggest that even if uncertainties remain about the precise implications of these results for human reproductive health, it is unlikely they are irrelevant. For aspiring parents wishing to have children, they indicate that cautionary steps to reduce exposures to these and related chemicals would make sense.

The ubiquitous use of the compounds studied in these experiments--and the fact that they can often be found in water supplies--means that while individual actions would likely be helpful, they will be insufficient to remove all relevant exposures. Hence more aggressive policies to encourage less use should be undertaken.