Low-Dose Agrochemicals and Lawn-Care Pesticides Induce Developmental Toxicity in Murine Preimplantation Embryos

Anne R. Greenlee; Tammy M. Ellis; Richard L. Berg

Disclosures

Environ Health Perspect. 2004;112(6) 

In This Article

Results

We used ethanol to prepare 10,000× stock solutions of 11 of 13 pesticides that were water insoluble. To test for solvent effects on preimplantation development, pronuclear embryos were incubated 96 hr in EMG without BSA supplemented with (n = 36) and without (n = 6) 0.1% ethanol (negative injury and solvent controls, respectively). This concentration represents the highest possible dose of ethanol in working dilutions of agrochemicals. After the incubation period, significant differences were not detected for the percentage developing to blastocysts (76.4 vs. 76.1%; p = 0.91), percentage of apoptosis (10.3 vs. 9.9%; p = 0.63), or mean cell number per embryo (111.2 vs. 109.7; p = 0.67) for ethanol controls and nonsupplemented controls, respectively.

Embryos were scored for development to blastocyst, mean cell number per embryo, and percentage of apoptosis after 96-hr incubation in controls, individual pesticides, and mixtures at low-dose concentrations based on 1× RfD values. Table 2 provide the weighted means of embryo developmental scores. Compared with the negative injury control treatments, 96-hr incubation of pronuclear embryos with the positive injury control (0.1 µg/mL o,p´-DDT) consistently reduced the percentage of development to blastocysts (all p ≤ 0.05) and increased the percentage of blastomeres undergoing apoptosis (all p ≤ 0.05). These findings are similar to those reported in two earlier studies (Greenlee et al. 1999; 2000).

Compared with the negative injury control treatments, incubating embryos with individual agrochemicals significantly increased the percentage of apoptosis for 11 of 13 chemicals tested, including dicamba, pendimethalin, 2,4-D, atrazine, chlorothalonil, mancozeb, diquat, metolachlor, ammonium nitrate, chlorpyrifos, and terbufos (all p ≤ 0.05). One herbicide (atrazine) and two insecticides (chlorpyrifos and turbufos) also reduced embryo development to blastocyst (all p ≤ 0.05). The fertilizer ammonium nitrate reduced mean cell number per embryo (p ≤ 0.0005). A reduction in embryo cell number was the only adverse effect noted (p ≤ 0.05) for the herbicide MCPP.

Mixtures, when compared with negative control treatments, reduced development to blastocyst or increased apoptosis, or had combined effects on blastocyst development and apoptosis. Mixtures formulated to represent preemergent herbicides (dicamba and pendimethalin) and postemergent herbicides (dicamba, 2,4-D, and atrazine) showed a pattern of injury similar to pesticides tested individually; for example, mixtures increased percentage of apoptosis in exposed embryos (all p ≤ 0.05) with no adverse effects on blastocyst development or embryo cell number. In contrast, mixtures formulated to represent groundwater contaminants (atrazine, metolachlor, 2,4-D, and ammonium nitrate), insecticides (chlorpyrifos, terbufos, and permethrin), and lawn-care herbicides (dicamba, 2,4-D, and MCPP) reduced blastocyst development (all p ≤ 0.05). The fungicide mixture (chlorothalonil/ mancozeb/ diquat) reduced development to blastocyst (p ≤ 0.05) and increased the percentage of apoptosis (p ≤ 0.005).

In summary, 12 of 13 individual chemicals and 6 of 6 mixtures at environmentally relevant concentrations induced developmental injury in preimplantation embryos. Only one agent, permethrin, had no measurable effects on developmental outcomes.

Photomicrographs of embryos representative of findings at the end of the culture period are shown in Figures 1 and 2 and correspond to results for control and pesticide treatments presented in Table 7 . Figure 1 shows development to blastocyst after incubating groups of 20-25 embryos with the negative (0.1% ethanol) and positive (0.1 µg/mL o,p´-DDT) injury controls, individual lawn-care herbicides (dicamba, 2,4-D, or MCPP), or the mixture of lawn-care herbicides (dicamba, 2,4-D, and MCPP). Approximately 70-80% of embryos incubated with the negative injury control (Figure 1A) or with the individual lawn-care herbicides (Figure 1C-E) developed to blastocyst, expanded blastocyst, or hatching blastocyst stages. Embryos at the blastocyst stage of development were characterized as having a thinned zona pellucida, a turgid blastocoele cavity, and a prominent inner cell mass (ICM). Expanded blastocysts showed further thinning of the zonae, with diameters larger than that of the blastocyst stage embryos. Hatching blastocysts exhibited partial protrusion of the embryo through the zona pellucida. Significantly fewer embryos incubated with the positive injury control (o,p´-DDT) (Figure 1B) or with the mixture of lawn herbicides (dicamba, 2,4-D, and MCPP) (Figure 1F) progressed to blastocyst (58-65%; all p ≤ 0.05). Residual embryos in o,p´-DDT, the positive injury control, and the herbicide mixture were often stalled at early cleavage stages (e.g., two-, four-, and eight-cell, and morula). Residual embryos in the negative injury control and individual pesticide treatment drops were frequently stalled at later cleavage stages (e.g., morula and preblast).

Photomicrographs shown in Figure 2 illustrate apoptosis results for embryos incubated in negative (Figure 2A) and positive (Figure 1B) injury controls and pesticide treatments (Figure 2C-F) detailed in Figure 1. The highest percentages of apoptosis were observed for embryos incubated with the positive injury treatment 0.1 µg/mL o,p´-DDT (Figure 2B) and for embryos incubated with the individual herbicides dicamba, 2,4-D, and MCPP (Figure 2C-E) (all p ≤ 0.05). The percentage of apoptosis for the herbicide mixture (Figure 2F) was not significantly different from the negative injury control (Figure 2A). Apoptotic nuclei were observed most commonly in the region of the ICM of blastocysts and stained yellow-green with TUNEL reagents.

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