Case Study Part 2: Probabilistic modelling of long-term effects of pesticides on individual breeding success in birds and mammals.

Long term exposure of skylarks to a fictitious insecticide and of wood mice to a fictitious fungicide were modelled probabilistically in a Monte Carlo simulation. Within the same simulation the consequences of exposure to pesticides on reproductive success were modelled using the toxicity-exposure-linking rules developed by R.S. Bennet et al. (2005) and the interspecies extrapolation factors suggested by R. Luttik et al. (2005). We built models to reflect a range of scenarios and as a result were able to show how exposure to pesticide might alter the number of individuals engaged in any given phase of the breeding cycle at any given time and predict the numbers of new adults at the season's end.

Case Study Part 1: How to calculate appropriate deterministic long-term toxicity to exposure ratios (TERs) for birds and mammals.

In the European Union, first-tier assessment of the long-term risk to birds and mammals from pesticides is based on calculation of a deterministic long-term toxicity/exposure ratio (TER(lt)). The ratio is developed from generic herbivores and insectivores and applied to all species. This paper describes two case studies that implement proposed improvements to the way long-term risk is assessed. These refined methods require calculation of a TER for each of five identified phases of reproduction (phase-specific TERs) and use of adjusted No Observed Effect Levels (NOELs) to incorporate variation in species sensitivity to pesticides. They also involve progressive refinement of the exposure estimate so that it applies to particular species, rather than generic indicators, and relates spraying date to onset of reproduction. The effect of using these new methods on the assessment of risk is described. Each refinement did not necessarily alter the calculated TER value in a way that was either predictable or consistent across both case studies. However, use of adjusted NOELs always reduced TERs, and relating spraying date to onset of reproduction increased most phase-specific TERs. The case studies suggested that the current first-tier TER(lt )assessment may underestimate risk in some circumstances and that phase-specific assessments can help identify appropriate risk-reduction measures. The way in which deterministic phase-specific assessments can currently be implemented to enhance first-tier assessment is outlined.

A new interpretation of avian and Mammalian reproduction toxicity test data in ecological risk assessment.

The long-term risks of pesticides to wildlife in the EU currently are assessed by comparing the lowest no-observed-effect concentration (NOEC) determined from the suite of endpoints measured in existing avian and mammalian laboratory reproduction tests with estimated exposure concentrations by calculating Toxicity to Exposure Ratios (TERs). Regulatory authorities experience difficulties when assessing long-term risks because of the lack of accepted methods to improve the ecological realism of exposure and toxicity estimates and understand risks at a population level. This paper describes an approach for interpreting existing avian and mammalian toxicity test data that divides breeding cycles into several discrete phases and identifies specific test endpoints as indicators of direct pesticide effects possible at each phase. Based on the distribution of breeding initiation dates for a species of concern and the dates of pesticide applications, this approach compares the phase-specific toxicity endpoint with the expected pesticide exposure levels during each of the breeding phases. The fate of each breeding attempt is determined through a series of decision points. The cumulative reproductive response of individuals in a breeding population based on this decision framework provides a means of examining the estimated risks over the course of the breeding season and deriving an overall metric of the impact of the pesticide on reproduction. Research needed to further improve the approach is discussed.

Toxicological assessment of biological pesticides.

The majority of pesticides are based on synthetic chemicals. Regulatory assessments are performed by comparing the findings in a range of routine toxicity studies, designed for testing chemicals, with estimates of exposures. Recently there have been significant moves towards developing natural/biological alternatives. Biological pesticides (those based on viable organisms) present the regulator with a different set of challenges to those raised by most chemical pesticides. The concerns associated with biological pesticides can vary greatly from one organism to another, requiring an almost case-by-case approach. The known toxicity of certain bio-molecules and the pathogenicity of certain organisms underlines the need for a risk assessment of biological pesticides. The main aspects of a health risk assessment are characterisation of the organism, infectivity, pathogenicity, sensitisation and production of toxic secondary metabolites. Obtaining information or data on these areas is not always easy as there are no widely accepted test schemes or protocols for organisms, though guidelines are being developed for the European Commission (EC). Predicting exposure following pesticidal use of an organism is made more complex if it multiplies or secretes toxic metabolites. Reliable data on effects (lack of) associated with naturally occurring (background) exposures can sometimes provide considerable reassurance. This paper describes the background to the proposed EC scheme, which has much in common with current UK practices, and presents three examples of biological pesticides which have been assessed under the existing UK procedures.

The use and limitations of current 'standard' toxicological data packages in the setting of acute reference doses.

To perform risk assessments for short term dietary intakes of pesticides it is necessary to determine an acceptable level of exposure from the available toxicity studies. This type of assessment is a relatively new development in regulatory toxicology and often the studies currently available are not optimal for such uses. The strengths and weaknesses of currently performed study types are presented. Consideration of the entire toxicity data base is essential.

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans: the risks to human health. A review.

1 PCDDs and PCDFs are ubiquitous and persistent in the environment. They are to be found in body tissues of both humans and animals. 2 The most extensively studied PCDD is 2,3,7,8-TCDD. It has been shown to produce a wide range of effects and is considered to be a (non-genotoxic) carcinogen in animals. 3 Studies into the mechanisms of toxicity so far reveal that there is involvement of a specific receptor (Ah), however further work is required to elucidate the mechanisms of the various effects. 4 Reports on a number of human exposures to PCDDs and PCDFs are described. Results from human epidemiological studies are difficult to interpret: there have been problems in methodology; there has been inadequate information on intake, and exposures have often been to mixtures of PCDDs and/or PCDFs together with other related compounds. 5 Many regulatory authorities faced with the problem of providing an index of risk from exposure to mixtures of PCDDs and PCDFs have employed the concept of 'TCDD equivalents'. 6 Whether or not PCDDs and PCDFs pose a significant human health risk at current levels of exposure they remain of considerable interest to the toxicologist.

Synergistic inhibition of [3H]muscimol binding to calf-brain synaptic membranes in the presence of L-homocysteine and pyridoxal 5'-phosphate. A possible mechanism for homocysteine-induced seizures.

L-Homocysteine and pyridoxal 5'-phosphate (PxyP) inhibited [3H]muscimol binding to freeze-thawed, Triton-treated calf brain membranes (containing high-affinity muscimol-binding sites: Kd 9.5 +/- 0.6 nM, Bmax 5.2 +/- 0.2 pmol/mg protein). The homocysteine--pyridoxal-5'-phosphate (Hcy-PxyP) thiazine complex had no effect. L-Homocysteine was found to be a partially competitive inhibitor, thus demonstrating an allosteric inhibition with Ki value of 1.96 mM for free receptor and Ki of 13 mM for receptor-muscimol complex. PxyP was shown to be a two-site pure competitive inhibitor of [3H]muscimol binding with cooperativity of PxyP binding such that Ki values for PxyP of 20 mM and 2.1 mM were found. L-Homocysteine and PxyP when added simultaneously to binding assays, caused a greater degree of inhibition than that observed at the same total specific concentration of either inhibitor alone. This synergistic inhibitory effect was shown to be due to a homocysteine-induced increase in the affinity of PxyP-binding sites. Three alternative models are suggested to explain the observed synergistic effects whereby it is proposed that PxyP and [3H]muscimol binding is mutually exclusive, while L-homocysteine with PxyP and L-homocysteine with muscimol, exhibit non-exclusivity. Non-linear regression analysis of binding data was undertaken in order to substantiate conclusions drawn from graphical procedures and in an attempt to ascertain which mathematical model best fitted the experimental data describing the synergistic inhibitory effects of L-homocysteine and PxyP. This synergistic inhibitory effect of PxyP and L-homocysteine on the post-synaptic gamma-aminobutyric acid receptor may provide a basis for explanation of the mechanism of homocysteine-induced seizures.

Hippocampal electrical activity and gamma-aminobutyrate metabolism in brain tissue following administration of homocysteine.

The concentration of gamma-aminobutyrate (GABA) and the activity of glutamate decarboxylase and GABA-transaminase were measured in extracts of mouse brain before the onset and during the course of generalized seizures induced by systemic administration of homocysteine thiolactone. The results indicate that whole brain GABA metabolism is unaffected by subconvulsive and convulsive doses of homocysteine at all stages of the generalized seizure. Electroencephalographic monitoring of rat brain electrical activity via hippocampal electrode implantation allowed the course homocysteine-induced seizures to be followed and afforded a means of quantifying such seizures.