Few bioassays are used according to Polish regulations:

1. Commercial kits incl. bioluminescence bacteria, any available Toxkit

2. Algae growth inhibition test

3. D. magna immobilization test

4. Lemna minor growth inhibition test

5. Fish acute test

Thanks. So, In your Country you have to utilize also the terrestrial organisms in the battery according with your legislation ? In Italy still do not, unfortunately.

Do you have any difficult applying the fish acute test?

For H14 there is no necessity to do terrestrial test, but for H5 and H6 classification Eisenia foetida test is required.

I did not do any test with waste so I can not tell you anything about specific difficulties in this topic. But from my experience and point of view the most confounding factors in fish testing are legal and ethical issues.

Thank you very much.

I think this paper should help you:

Pandard, P., Römbke, J., 2013. Proposal for a “Harmonized” strategy for the assessment of the HP 14 property. Integr Environ Assess Manag 9, 665–672. doi:10.1002/ieam.1447

See also: Pandard, P., Devillers, J., Charissou, A.-M., Poulsen, V., Jourdain, M.-J., Férard, J.-F., Grand, C., Bispo, A., 2006. Selecting a battery of bioassays for ecotoxicological characterization of wastes. Science of The Total Environment 363, 114–125. doi:10.1016/j.scitotenv.2005.12.016

Thank you very much.

I'll take these papers in consideration.

You're welcome :)

But basically, wastes can be assessed (not according to regulations) using aquatic organisms (bacteria V. fischeri luminescence, algae P. subcapitata growth, crustacean D. magna mobility/reproduction, rotifer B. calyciflorus reproduction, zebra-fish D. rerio mortality) and terrestrial organisms (higher plants root elongation/biomass, earthworm E. fetida avoidance/mortality/reproduction and collembola F. candida avoidance/mortality/reproduction).

Thanks again.

If I can ask you...what do you think about embryo toxicity test of danio rerio applied on waste?

Well, cyto-toxicity for the assessment of organic wastes was already proposed by some authors on fish cell-lines (i.e. Delgado, M., Imperial, R.M. de, Alonso, F., Rodríguez, C., Martín, J.V., 2013. Ecotoxicity Bioassays on Leachates from Poultry Manure. Bull Environ Contam Toxicol 90, 401–404. doi:10.1007/s00128-012-0936-9).

I'm not really expert in fish toxicity (much more on aquatic and terrestrial invertebrates), but here you may find useful informations: http://www.oecd.org/chemicalsafety/testing/36817242.pdf

For complementary informations about H14 assessment, have a look at these papers:

- Wilke, B.-M., Riepert, F., Koch, C., Kühne, T., 2008. Ecotoxicological characterization of hazardous wastes. Ecotoxicology and Environmental Safety 70, 283–293. doi:10.1016/j.ecoenv.2007.10.003

- Pablos, M.V., Fernández, C., del Mar Babín, M., María Navas, J., Carbonell, G., Martini, F., García-Hortigüela, P., Vicente Tarazona, J., 2009. Use of a novel battery of bioassays for the biological characterisation of hazardous wastes. Ecotoxicology and Environmental Safety 72, 1594–1600. doi:10.1016/j.ecoenv.2008.12.016

- Stiernstroem, S., Hemstroem, K., Wik, O., Carlsson, G., Breitholtz, M., 2009. Methodology for classification of the H14 criterion according to the directive 2008/98/EC on waste. Proposal of a biotest battery for the classification of hazardous waste. Ecotoxicological testing with bacterium, algae, crustacean and fish embryo; Metodik foer klassificering av H14-kriteriet i Avfallsfoerordningen. Foerslag till biotestbatteri foer klassificering av farligt avfall. Ekotoxikologisk testning med bakterie, alg, kraeftdjur och fiskembryo.

- Weltens, R., Vanermen, G., Tirez, K., Robbens, J., Deprez, K., Michiels, L., 2012. Screening tests for hazard classification of complex waste materials – Selection of methods. Waste Management 32, 2208–2217. doi:10.1016/j.wasman.2012.05.013

I hope you will find what you need.

Also, considering your second question "Do you prepare the leachate with distilled water or with the organism test water?", from what I know, leachates are obtained directly from the waste (water and/or solvent), which is diluted in bioassays medium to obtain a concentration range (something like from 0% of sample to 90% of sample, as 100% means no use of bioassays medium...)

Have a good week end

Pierre, Thank you very much for your answers.

Yes the leachate is obtained from waste with deionized water but when I tested sample at 100%, following ISO, I think is not quite good for organisms that need some microelements present instead in their medium.

Leachate generation is a major problem for municipal solid waste (MSW) landfills and causes significant threat to surface water and groundwater. Leachate is the liquid that has percolated through solid waste. Leachate management is one of, if not the most significant long-term, recurring cost for landfill care.Contaminants in the  buried refuse may result from the disposal of industrial waste, ash, waste treatment  sludge,  household hazardous wastes, or from normal waste decomposition. If uncontrolled, landfill leachate can be responsible for contaminating ground water and  surface water.

The composition of leachate varies greatly from site to site, and can vary within a

particular site.A designed lining system, which ensures low-permeability limit the movement of  leachate into ground water. Liners are made from low-permeability soils (typical  clays) or synthetic materials (e.g. plastic). Landfills can be designed with more than  one liner, and a mix of liner types may be used. Physical treatment, Chemical treatment and Biological treatment methods are available which can find solutions of ecotoxicological risk managment.