Effects of agricultural pesticides on the health of Rana pipiens frogs

The balance of natural ecosystems is almost certainly being disturbed by human modification of the natural environment. One such modification is the use of pesticides, which may leak into the surrounding soil and water and influence the surrounding ecosystems. Prior lab results have shown that pesticides could affect the health status of amphibians. To verify if similar results would be seen in natural populations, Cristin et al. (2013) sampled leopard frogs at three contaminated sites and two reference sites and used size, weight, spleen cellularity, phagocytic activity, and other measurements to examine the differences between frogs collected from contaminated sites and reference sites. The results of the study show that the juvenile frogs exposed to agripesticides are smaller in weight and length. It was also found that the number of active phagocytes, cells that protect the body by ingesting potentially harmful foreign particles and bacteria, was significantly reduced in exposed frogs. Thus pesticide use may inhibit amphibian growth and immune system response. —Kahea Kanuha

                  Christin, M.S., Menard, L., Girous, I., Marcogliese, D.J., Ruby, S., Cyr, D., Fournier, M., Brousseau, P. 2012. Effects of agricultural pesticides on the health of Rana pipiens frogs sampled from the field. Environmental Science and Pollution Research published ahead of print September 21, 2012, doi: 10.1007

                  Cristin et al. began by selecting five study sites: three contaminated sites and two reference sites along the tributaries of the St. Lawrence River in Monteregie, Quebec, Canada. The contaminated sites were directly adjacent to agricultural land and were therefore exposed to pesticide runoff. The reference sites included a conservation wetland and a wetland within a rural park, with managed landscape and human activity nearby. Pesticide concentrations were measured through collections of surface water samples. Repeated sampling was necessary since pesticide concentrations may fluctuate considerably with rain patterns and pesticide application time in the surrounding crop fields. For each site, temperature, conductivity, pH, and nitrate concentrations were recorded.
                  Juvenile R. pipiens were captured at the end of July and the beginning of September at each of the five sites. Overall health of the frogs was quantified through measurements of length and weight. The body index, or ratio of weight to length, was used rather than the measurements themselves in subsequent analyses.
To determine immune response impairment, the authors examined the viability of splenocytes, cellularity, and phagocytosis. Splenocytes are white blood cells produced in the spleen, and play an important role in immune system response. The spleen of each animal in the study was removed and cell suspensions were then prepared. Cellularity is the number and type of cells present in a given tissue. Viability, essentially the amount of living cells, was measured to ensure cell extraction methods were effective and that there were enough living cells to proceed with phagocytosis analysis. Both cellularity and splenocyte viability were determined microscopically after trypan blue dye exclusion, which stains dead cells blue.
Phagocytes are cells that ingest harmful foreign particles to protect the body and constitute the first line of defense against infectious microorganisms. Phagocytic activity of splenocytes was determined by introducing fluorescent bacteria to the spleen cells, incubating the cells, and then collecting the fluorescence emission. Higher fluorescence emissions indicate higher bacteria count and thus less phagocytic activity.
The results indicate that frogs living in sites exposed to pesticides runoff are smaller in length and weight
                  In both July and September, frogs living in agricultural regions had significantly lower body indices than frogs sampled in reference sites. In addition, there were no significant differences of body indices between the two reference sites. Spleen cellularity results show significant decreases in the number of splenocytes for frogs sampled in the three contaminated sites. However, in September one of the reference sites showed a number of splenocytes six times higher than the other reference site. Viability for all groups of frogs captured was over 70%, indicating the splenocyte suspensions were suitable to be used in the phagocytosis assay because there were enough living cells. The phagocytosis results for frogs sampled in July show a significant decrease in phagocyte numbers for four of the five contaminated sites. The September results show a significant decrease in two of the contaminated sites. In both months there was no significant difference between the reference sites.
                  It has already been observed that pollutants can reduce tadpole growth, but the exact way in which contaminants impair growth is not fully understood. Contaminated environments may hasten a tadpole’s metamorphosis in order to escape from a poor growing environment, which leads to a smaller size. However, the smaller size of frogs sampled from contaminated sites in this study may be due to other reasons, such as physiological effects of contaminants on growth or food limitation due to low food quality. The specific interactions between pesticides and the immune system also warrant further investigation in order to fully understand the impact of agricultural practices on aquatic systems.

This study has shown that juvenile frogs exposed to agripesticides are smaller and have compromised immune systems compared to frogs captured in reference sites. Further studies on almost all aspects of the relationship between agrochemicals and ecosystem response are necessary to more fully understand the complex consequences of human actions.

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