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Ciao Michela, 

I never worked with your test organism, but here follows some information on the the toxity that applies for most of aquatic species.

EDTA has potential to bind metals, and therefore affect the free ion concentration. As the free ion concentration (bioavailable metal) is normally accepted as the one responsible for most of toxic effects, adding EDTA has potential to affect toxicity.  Naturally, how much it would affect the toxicity of Zn would depend on the water chemistry of your experimental media. In other words, it is difficult to say whether you would be able to measure the effect of EDTA without knowing the concentrations of EDTA, Zn treatments, and other cations, anions, and ligands in your media. While the effect of EDTA is always there, the question on whether you would sense it depends on your experimental setup.

Regarding the comparison to natural water, again it depends on the EDTA concentrations and the binding capacity of the EDTA you are using. 

I would be careful to add EDTA to metal exposures, especially if you want them to be comparable to natural waters. There are other metal complexing agents that could be more useful for that, as humic acids for instance.

I hope it helped

abel ;)

You should avoid adding chelating agent when testing metals. This is a citation from OECD 201 guideline: "Modification of the growth media may be necessary for certain purposes, e.g. when testing metals and chelating agents".

Take care when making algae test in natural waters. Due to limited amount of nutrients in such medium you may not be able to get any growth of algae in controls. Also proper sterilization of natural waters must take place to ensure that no grazing zooplankton and no wild algae species is present. 

You could try understanding your system (i.e. speciation of Zn in your media and growth of algae) with the following treatments couldn't you: 1) OECD 201 nutrient solution prepared in you ultrapure or distilled water, 2) NS+UPwater+spiked Zn, 3) NS+your natural water with Zn, 4) NS+a relevant reference natural water without Zn (this can be tricky to find though...). You can also just simply leave the Na2EDTA*2H2O out of your NS. Yet here are other sources of Na in the NS. Or prepare treatments with nutrient solutions containing Na2EDTA*2H2O and without it and use both in all of your treatments to see how limiting factor it can be for the algae growth in you treatments (so duplicate).

Yes, absolutely, EXCESS EDTA will chelate metals and reduce bioavailability, and hence toxicity.

But EDTA is required in the algal media to assist with the uptake of Fe. Exclusion of EDTA completely with result in poor growth.

The trick is the balance the molar concentrations of Fe and EDTA, so that there is minimal excess EDTA to bind other metals.

Na2EDTA x 2 H2O  = 0.05 mg/L in final media should do it (all other components as OECD 201).