Professional Interest Groups :: Dung Organisms Toxicity Testing (DOTTS)

Dung Organism Toxicity Testing Standardisation Group (DOTTS)

There is currently an initiative to harmonize testing requirements for veterinary medicines, known as the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Products (VICH). The parties to this process are the European Union (EU), Japan, and the USA, with Australia/New Zealand and Canada as observers.
During discussions of the VICH Ecotoxicity/Environmental Impact Assessment Working Group it was noted that there are currently no internationally recognized guidelines for laboratory testing for effects of veterinary medicines on dung flies and dung beetles.

Through discussions between European Union (EU) regulators, Australian industry, government and research representatives, and EU contract laboratories an initiative was begun to develop and ring-test toxicity test methods for dung beetles and dung flies. An inaugural meeting of interested parties took place on 25 and 26 February 2002, with representatives from Australia, Canada, Denmark, France, Belgium and the UK (today the group covers most of the EU, North America, South Africa, and Asia/Pacific). One outcome of this meeting was the formation of the Dung Organism Toxicity Testing Standardisation (DOTTS) Group. A second meeting of the group was organized in Hamburg in May 2003. The activities of the group are described below in detail.

The aims of the group are as follows:

• exchange of information about testing the effects of veterinary drugs on dung organisms;
• development of test protocols for toxicity testing with dung flies and dung beetles; and
• performance of ring tests with dung flies (starting in 2003) and dung beetles (starting in 2004) in order to standardize and validate the test protocols.

In December 2002 DOTTS became affiliated with SETAC Europe. As such, the group abides by the scientific principles of SETAC, and acts in the interests of government regulators, the veterinary pharmaceutical industry and research institutions (e.g. universities or contract laboratories). It is expected that within the next three years DOTTS will provide
OECD with a draft of up to four test protocols (two each for dung flies and dung beetles, covering temperate as well as tropical regions).

Development of a test with dung flies

A standardized bioassay procedure for Musca autumnalis De Geer (Diptera: Muscidae), Musca vetustissima Walker (Diptera: Muscidae) and Scathophaga stercoraria L. (Diptera: Scathophagidae) has been proposed by J. Hughes, K. Wardhaugh, B. Rosenkranz and K. Floate. The protocol is designed to estimate the toxicity of a test chemical to the dung
dwelling life stages of dung-dependant dipteran species. A positive control and negative controls (solvent and untreated) are included as comparisons. In this test, insects are exposed under controlled but severe (“worst case scenario”) conditions. The test chemical is mixed with bovine faeces, to which larvae are added. The impact of the test chemical on
maturation of the larvae to adults is assessed. Test dung is not changed during the study. The description and performance of this test follows current requirements of GLP (Good Laboratory Practice).

Musca autumnalis, Musca vetustissima and Scathophaga stercoraria are considered to be suitable indicator species for estimating the acute toxicity of parasiticides on dung dependant diptera for the following main reasons:

Collectively, the species cover a wide geographic range. M. autumnalis and S. stercoraria are widespread in Europe, Asia, Africa and North America. M. vetustissima is found throughout Australia and is closely related to the ‘narrow fronzed’ strain of M. sorbens, which is widespread in south-east Asia. All three species are dung-dependant. All three species are multi-voltine, do not undergo obligate diapause and are easy to culture. All three species have a short life-cycle which makes it possible to determine acute effects on survival in the laboratory.

Development of a test with dung beetles

As in the case of dung flies, two test protocols covering paracoprid beetles and endocoprid beetles are under development. Paracoprid beetle species tend to be found in warmer, drier climates such as Australia, South Africa, the Mediterranean and Asia, whereas Endocoprid beetles are more typical of temperate areas such as large parts of Europe and North America.

Onthophagus taurus and Euoniticellus fulvus are opportunistic, multivoltine paracoprid beetles with a generation time of about 6 weeks at 26oC. When fed on good quality dung, females of both species attain sexual maturity in 1-2 weeks and will lay 1-2 eggs daily for four or more weeks. Egg to adult emergence will occupy about 4-6 weeks. Both species are therefore well suited for use in tests of excreted residues of veterinary parasiticides. A standardized bioassay procedure has been proposed by K. Wardhaugh and agreed within the DOTTS Group. This is based on methods used in the scientific literature that have been modified to make them more practical for a contract laboratory environment, in particular by reducing the amount of space needed to do the tests. Sexually-mature beetles are exposed to treated dung and the test investigates brood-ball production, adult emergence from brood balls and subsequent survival. This protocol has already been submitted to OECD as a possible future guideline, once ringtesting and protocol development is complete. A beetle culture has successfully been established at a contract laboratory in the UK and an initiative has been put in place to start some ring-testing in Australia. Other DOTTS laboratories are encouraged to join in with this ring-testing.

The second test uses species of the genus Aphodius (Coleoptera: Aphodiidae). The German Federal Environmental Agency (UBA, Berlin) is sponsoring the development of a test using the species A. constans. The following milestones have been achieved:

• development of a laboratory rearing method for this species;
• laboratory tests comparing dung spiked with parasiticdes with dungvoided by cattle treated with parasiticides;
• clarification of test methodology (e.g. homogenization of chemicals in dung substrate), and
• formulation of a draft test guideline according to OECD standard.

In parallel, other species of this family are being investigated to determine if they can also be used as test organisms.

Outlook

It is expected that the preparation for the ringtest with dung flies will be finished within 2003. Assuming that the ringtest can be finalized by the end of 2004 a draft guideline will be published in early 2005. The development of such a draft for the beetle test will probably take one year longer.

Acknowledgements

This work is supported by the German Federal Environmental Agency (Berlin); Project No. 202 67 428.