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EC number: 222-093-9
CAS number: 3344-18-1
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.
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
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.
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
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).
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).
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).
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
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).
3.1 Stability constants of citric acid
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).
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.
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
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.
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.
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.
the hazard assessment for this substance based on the properties of
citric acid is valid.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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