Terrestrial Ecotoxicity Tests in New Zealand

• Soil tests
• Plant tests
• Soil micro-organism tests
• Soil invertebrate tests
  • Earthworm tests
  • Woodlouse tests

Benzene, Toluene, Ethyl Benzene & Xylene (BTEX) Ecotoxicity Tests

Some pilot studies were carried out to determine ecotoxicity values for BTEX to soil invertebrates and plants using standardised methodologies. However, under the standardised conditions prescribed by the testing methodologies we were unable to obtain consistent soil concentrations, as these compounds were rapidly volatilised from the test soil.  Further explanation and background is given here.

Soil Tests

A New Zealand Templeton silt loam (5% organic matter, pH = 6.2) was used for our plant, earthworm, and woodlouse tests, thus allowing standardisation of conditions across the different tests. The soil was 'spiked' with a range of concentrations of the test compound. A 'standard' soil is preferable to the OECD 'artificial' soil recommended in the earthworm guideline (OECD 1984b) as it allows extrapolation to the field, and will also support the plant tests. 

Details Regarding Soil Test Methodology

Soil tests were conducted using a Templeton silt loam soil with the following characteristics:

This standard laboratory soil was field collected, air dried and sieved through a 5 mm2 sieve, and stored undercover.

Copper, Chromium and Arsenic (CCA) Ecotoxicity Tests

Soils were spiked with analytical grade CuSO4, Na2Cr2O7 and As2O5 (common contaminants at timber treatment sites). To ensure even application of metals throughout the soil, working solutions of metal salts dissolved in de-ionised water were added to the soil at a rate equal to or less than 5 ml per 100 g fresh weight so that 60% MWHC (maximum water holding capacity) was not exceeded. Metal solutions were added to 2 kg batches of dry soil and were thoroughly mixed using a Kenwood mixer with a dough K-blade set on 1 for 30 seconds. After the addition of metals, grass meal was added to the soil for the soil invertebrate assays.

An initial range finder test using 0 - 2400 mg metal ion/kg soil dry weight was conducted to determine the approximate toxicity of the metals. This range represents the range of metals concentration likely to be found in soils at CCA contaminated sites. The test concentration range was selected for the following definitive test according to the results of the range finder test. The metal concentrations used are described in the results tables.

Soil for the invertebrate tests was allowed to rest at 20^oC for 1 week as specified in the OECD guidelines. For the plant tests, seeds were planted within 24 hours of incorporation of test compound as specified in the OECD guidelines. Prior to commencement of tests, soil samples were taken for residue analysis.

There is no universal standard plant test used by testing agencies, although the OECD guideline No. 208 (OECD, 1984a) is often quoted for soil contaminants and the USAEPA OPPS guidelines are utilised specifically for plant protection products. The plant testing conditions are based on a combination of the OECD guideline, and a comprehensive guideline published by the American Society for Testing and Materials (ASTM 1994, # E1598-94).

Plant tests

Soil micro-organism tests

Microorganisms are important for the breakdown and transformation of soil organic matter, with many species contributing to different aspects of soil fertility. It is not possible to assess the impacts of soil contaminants on individual microorganisms in soils and extrapolate these effects to the whole community. 

For indigenous soil microorganisms, our ecotoxicity test assessed the functional integrity of microbial nitrogen cycling by measuring nitrogen mineralisation from standardised plant material. Our test was derived from the draft OECD guideline (OECD 1996) for testing the effect of chemicals on microbial processes based on the recommendations of the European and Mediterranean Plant Protection Organization (EPPO 1994).

As recommended by the soil microbial tests workgroup at the workshop on standardised test methods for soils (Fairbrother et al. 1999), toxicity in natural soils containing indigenous microbial communities was examined.  We selected three control subsoils representative of metal contaminated sites in New Zealand.  However, we were unable to obtain satisfactory and reproducible dose-response relationships between nitrogen mineralisation and exposure to metals using this microbial test method, in all except for one test.  We are therefore unable to report the ecotoxicity values for micro-organisms in New Zealand soils.

The OECD guideline for toxicity testing in earthworms No. 207 (OECD, 1984b) utilises adult Eisenia fetida selected for ease of production and maintenance in culture. This earthworm, however, is not a natural soil-dwelling species, and its occurrence in the wild is limited to sites rich in organic matter. Experts have recommended enhancing the ecological relevance of the standard earthworm test by using indigenous soil-dwelling species such as Aporrectodea caliginosa.  However, it is said to be difficult to breed and handle in the laboratory (Kula & Larink 1998). We have not found this to be the case, and are currently breeding and maintaining this species with relative ease. Aporrectodea caliginosa is the most common earthworm in New Zealand pastures and inhabits the topsoil, thus making it a far more valuable test species than E. fetida. 

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