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Toxicity to aquatic algae and cyanobacteria

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Description of key information

In accordance with Section 2 of REACH Annex XI, the study does not need to be conducted because an assessment of toxicity to aquatic algae and cyanobacteria is technically not feasible because of the substance's capacity to complex essential nutrients present in the test media, rendering them unavailable for uptake.

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In accordance with Section 2 of REACH Annex XI, the study does not need to be conducted because an assessment of the toxicity to aquatic algae andcyanobacteriais technically not possible due to substance'scomplexingproperties of essential nutrients present in the test media.


It is a functional property of citrates to form stable complexes (ligands) with metal ions. In algal toxicity tests essential nutrients will thus be bound to the citrates according to the Ligand binding model[1]. In algal growth medium some metals form strongly-bound complexes and others form weakly-bound ones. The citrates possess multiple metal-binding capacities, and pH will affect the number of binding sites by altering the ionisation state of the substance. However, the citrates ionisation is extensive regardless of the presence of metals (PFA 2010).


The citrate-metal complexes may be very stable due to the formation of ring structures ("chelation"). This behaviour ensures that the citrates effectively bind and hold the metals in solution and renders them biologically less available As a result when a trace metal is complexed, its bioavailability is likely to be negligible. However, there is no evidence of severe toxicity from metal complexes of the ligands (PFA 2010).


In algal growth inhibition tests, complexation of essential trace nutrients (includingFe and Zn) by citrate substances can lead to inhibition of cell reproduction and growth. Guidelines for toxicity tests with algae typically do not describe procedures for mitigating against this behaviour. For example the standard OECD Guideline 201, describing the algal growth inhibition test, only specifies that the “chelator content” should be below 1 mmol/l in order to maintain acceptable micronutrient concentrations in the test medium (PFA 2010).


OECD guidance on the testing of difficult substances and mixtures (OECD, 2000) does include an annex describing “toxicity mitigation testing with algae for chemicals which form complexes with and/or chelate polyvalent metals”. The procedure is designed to determine whether it is the toxicity of the substance or the secondary effects of complexation that is responsible for any observed inhibition of growth. It involves testing the substance in its standard form and as its calcium salt in both standard algal growth medium and in medium with elevated CaCO3hardness. Calcium is non-toxic to aquatic organisms and does not therefore influence the result of the test other than by competitively inhibiting the complexation of nutrients. By increasing the calcium content it may be that the nutrient metals are released from their complexed form although this may not always apply. The outcome of the test however only determines whether nutrient complexation is the cause of apparent toxicity and does not determine the inherent toxicity of the test substance for the reasons explained by the Ligand binding model (PFA 2010).

The magnitude of the stability constants depends on the properties of the metal and also of the ligand, in respect of the type of bonding, the three dimensional shape of the complexing molecule, and the number of complexing groups.

The complexation strengths expressed as the log10of the stability constant are enlisted mainly in the handbook from Furia (1972), and in other references (Martell 1989, Martin 1994).

Table 3.1          Stability constants of citric acid


Stability constant 




























Calculation based on the known stability constants shows that even where the OECD-recommended approach to add additional calcium to the test media is used, the complexation properties of these ligands means that key nutrients would still be complexed by the citrates in preference to complexation of calcium and magnesium, and therefore the calcium complex (most representative of the environmental species) can never be maintained in the test medium in the presence of other key nutrient ions such as Fe, Zn and Cu. The resulting complexed nutrients will almost certainly not be bioavailable to aquatic plants and this can result in inhibited algal growth. Growth inhibition viathis mechanism is a secondary effect and does not reflect the inherent toxicity of the test substance (PFA 2010).


A study designed to ensure adequate levels of bioavailable nutrients with either of the citrates would result in the actual substance tested being a citrate-Fe complex. Under conditions where iron is readily available to counteract the effects of nutrient complexation it is unlikely that the substance will have a negative effect on algal growth. The nutrient complexing behaviour of citrate substances therefore renders testing to determine their intrinsic toxicity to algae impractical. The available evidence suggests that toxic effects observed in the tests are a consequence of complexation of essential nutrients and not of true toxicity.


It is thought that this nutrient complexing phenomenon will not have an impact on algal growth in a fluid matrix such as natural waters where nutrients are available in a non-limited manner. Infinite


[1]Ligand’ is a general term used to describe a molecule that bonds to a metal; in the present case the citrate can form several bonds and the resultant chelated complex can be a very stable entity. It is possible that two molecules could bind to the individual metal, or that one molecule could bind two metals. In dilute solution a 1:1 interaction is the most probable. To simplify discussion, the ligand is considered to be able to form a strongly-bound complex with some metals, and a more weakly-bound complex with others.


However some reliable measured data is available indicating that notwithstanding the parent substance citric acid is of low toxicity. The 8 day toxicity threshold (TT) value - defined as a value between the NOEC and the LOEC, for citric acid to Scenedesmus quadricauda was 640 mg/L and the extrapolated NOEC was estimated at 425 mg/L.

It is possible to read-across citric acid to trisodium dicitrate since information available in the public domain on tests carried out on other salts of this metal indicates that the sodium ions are not expected to contribute to the toxicity of the substance. Additionally, the substance will dissociate when in solution, so the test organisms will be exposed to the citrate and the metal ions separately.

Therefore, the hazard assessment for this substance based on the properties of citric acid is valid.