General

The aim of this study was to provide EFSA with information relating to assessment of the risk to amphibians posed by pesticide exposure. In the first part of the study the European amphibian species associated with agricultural habitats were identified with the aim of collating information for representative species such as body size and life-cycle. Also collated were the results of studies of amphibians in European agricultural habitats to provide information on activity in areas where they may be at risk of exposure to pesticides.

Atrazine, the most widely used herbicide in the United States, has been shown in several studies to be an endocrine disruptor in adult frogs. Results from this study indicate that atrazine also functions as an immune disruptor in frogs. Exposure to atrazine (21 ppb for 8 d) affects the innate immune response of adult Rana pipiens in similar ways to acid exposure (pH 5.5), as we have previously shown. Atrazine exposure suppressed the thioglycollate-stimulated recruitment of white blood cells to the peritoneal cavity to background (Ringer exposed) levels and also decreased the phagocytic activity of these cells. Unlike acid exposure, atrazine exposure did not cause mortality. Our results, from a dose–response study, indicate that atrazine acts as an immune disruptor at the same effective doses that it disrupts the endocrine system.

An injection study and a field study were used to investigate the hypothesis that environmental xenobiotics have the potential to alter the immune function of northern leopard frogs (Rana pipiens). Three assays, IgM-specific antibody response to keyhole limpet hemocyanin linked to dinitrophenyl (KLH-DNP), zymozan induced chemiluminescence (CL) of whole blood and the delayed-type hypersensitivity (DTH), were used to assay humoral, innate and cell-mediated immune endpoints. Sublethal doses of DDT (923 ng/g wet wt), malathion (990 ng/g wet wt), and dieldrin (50 ng/g wet wt) were used in the injection study. In all pesticide-injected groups, antibody response was dramatically suppressed, DTH reactions were enhanced, and respiratory burst was lower. When the order of administration of pesticides and antigens was reversed, no differences in immune function between the control and dosed groups were apparent, indicating that frogs exposed to pathogens prior to pesticide exposure can still respond. A field study found significant differences in immune function between frog populations in pesticide-exposed and pesticide-free locations. The antibody response and CL were suppressed and the DTH enhanced in frogs from Essex County (ON, Canada).
Overall, the results suggest that exposure to these pesticides can cause both stimulatory and suppressive immune changes in adult frogs and is doing so in wild populations.

Recently, a decrease in the populations of marine toads (Bufo marinus) and whistling frogs (Eleutherodactylus johnstonei and E. gossei) has been noted on the island of Bermuda. In the current study, we investigated whether this decline was related to altered immune functions. During August 1998, a significant proportion (25%) of the toads exhibited deformities. Analysis of soil samples revealed presence of chlorinated pesticides and polychlorinated biphenyls (PCBs). Spleen cells from toads collected from more polluted areas of Bermuda exhibited a decrease in B-cell proliferative response to lipopolysaccharide when compared to the responsiveness of B cells from toads collected from less polluted areas. In contrast, the T-cell responsiveness to mitogens in these two groups was not significantly altered. Histological examination of major parenchymatous organs in marine toads and whistling frogs showed alterations in hepatic and splenic morphology, indicative of exposure to toxicants. Together, the current study suggests that the immune functions of toads from contaminated sites of Bermuda are significantly altered, which may contribute to the decline in their population.

Outbreaks of infectious diseases in honey bees, fish, amphibians, bats and birds in the past two decades have coincided with the increasing use of systemic insecticides, notably the neonicotinoids and fipronil. A link between insecticides and such diseases is hypothesised. Firstly, the disease outbreaks started in countries and regions where systemic insecticides were used for the first time, and later they spread to other countries. Secondly, recent evidence of immune suppression in bees and fish caused by neonicotinoids has provided an important clue to understand the sub-lethal impact of these insecticides not only on these organisms, but probably on other wildlife affected by emerging infectious diseases. While this is occurring, environmental authorities in developed countries ignore the calls of apiarists (who are most affected) and do not target neonicotinoids in their regular monitoring schedules. Equally, scientists looking for answers to the problem are unaware of the new threat that systemic insecticides have introduced in terrestrial and aquatic ecosystems.