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EC number: 267-956-0
CAS number: 67953-76-8
Endpoint waived on the basis
that the study is technically unfeasible. The essential nutrients
present in the test medium will be complexed by the phosphonates and as
a result the test organisms will be exposed to phosphonate-metal
complexes. Adverse effects seen in the studies are therefore likely to
be theresult of nutrient complexation rather than a reflection of the
true toxicity of the test substance
Several algal studies are available with HEDP acids and salts. The
effects of HEDP observed in tests with algae are likely to be a
consequence of nutrient availability limitations caused by
complexation and not true toxicity (see the discussion below). The
test results that are available cannot therefore be taken into
account to assess the toxicity of HEDP. They have been included as
supporting information to show the variability in determined EC50
and NOEC values and to illustrate the issues with testing
substances that exhibit chelating behaviour. The available evidence
suggests that toxic effects observed in the tests are a consequence
of complexation of essential nutrients and not of true toxicity.
A study with DTPMP (TNO, 1996) has also been included as supporting
evidence that observed effects are due to the consequence of
complexation of essential nutrients (see below for details). The
test demonstrates effects of iron-DTPMP complex to algae, not any
effects of the free substance.
It is therefore concluded that the nutrient complexing behaviour of
phosphonate substances renders testing to determine their intrinsic
toxicity to algae impractical. However, there is no evidence of
severe toxicity from metal complexes of the ligands.
Information requirement: Growth
inhibition study with algae / cyanobacteria
technically not feasible
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
and cyanobacteria is technically not possible due to the substance's
complexing properties of essential nutrients present in the test
The effects of HEDP observed in tests with algae are likely to
be a consequence of nutrient availability limitations caused by
complexation and not true toxicity (see below). The test results that
are available cannot therefore be taken into account to assess the
toxicity of HEDP.
Nutrient complexation in algal test medium
It is a functional property of phosphonate substances that they
form stable complexes (ligands) with metal ions. In algal toxicity
tests essential nutrients will thus be bound to the
phosphonates according to the Ligand binding model.
In algal growth medium some metals form strongly-bound complexes and
others form weakly-bound ones. The phosphonates possess multiple
metal-binding capacities, and pH will affect the number of binding
sites by altering the ionisation state of the substance. However, the
phosphonate ionisation is extensive regardless of the presence of
metals (Girling et al. 2010).
The phosphonate-metal complexes may be very stable due to the
formation of ring structures ("chelation"). This behaviour ensures
that the phosphonic acids 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 (Girling et al. 2010, SIAR 2004). However, there is
no evidence of severe toxicity from metal complexes of the ligands
(Girling et al. 2010).
In algal growth inhibition tests, complexation of essential trace
nutrients (including Fe, Cu, Co, and Zn)
by phosphonate substances can lead to inhibition of cell reproduction
and growth. Guidelines for toxicity tests with algae do not typically
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 (SIAR 2004).
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 CaCO3 hardness. 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 (SIAR 2004). 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
(Girling et al. 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 SIAR provides two tables of stability
constants (effectively the strength of the complexation), one from
Lacour et al. (1999) and one from Gledhill and Feijtel (1992).
The Gledhill and Feijtel constants show a range of values for
important divalent metal ions, cited as having been obtained from
Monsanto internal reports (Owens, 1980). They show that ATMP, HEDP and
DTPMP are strong complexing agents, with stability constant values
ranging from 6 to 24 (Log10 values), as presented in the
Table: Stability constants of phosphonates.
a - The complexation constant for phosphonates with iron (III)
has been estimated by TNO (1996a) to be around log K = 25.
b – In the absence of experimental data, the stability constants
of BHMT complexes has been estimated as the mean of the stability
constants for each metal ion as measured with the structural analogues
DTPMP and HMDTMP.
The complexation constant for phosphonates with iron (III) has been
estimated by TNO (1996a) to be around log K = 25 (Girling et al. 2010).
All the algal toxicity studies available for phosphonates that have used
standard and non-standard test conditions are presented in Girling et
al. (2010). The studies show a large variation of toxicity for these
substances sharing similar physico-chemical properties, with reliable EC50
varying from 0.1 to 450 mg/l.
The most refined study to date is the DTPMP study undertaken by TNO
laboratories (1996) where concentrations of Cu, Co and Zn, were
increased in the medium in line with their complexation strength (Cu up
to 30 times, Co up to 30 times and Zn up to 300 times). When Fe was also
added up to 300 times the guideline concentration no toxic effects were
seen at the highest tested concentration (96h ErC50 equivalent
to >10 mg/L). The increased amounts of Fe meant that complex iron-DTPMP
bonds were formed, leaving the four nutrients free for algal uptake. The
test demonstrates effects of iron-DTPMP complex to algae, not any
effects of the free substance. The media concentration of Fe in the
study is a highly unlikely scenario in a true environmental exposure,
where Ca and Mg are likely to be more readily available but are also
more weakly complexed. Where essential nutrients with stronger binding
capacity are present, such as Cu, Co, Zn and Fe, the phosphonates will
preferentially bind to these nutrients leaving the Ca and Mg free.
In Springborn Laboratories (1992) the mitigation procedures
suggested in the OECD guidance on testing difficult substances (2000)
were adopted when testing with HEDP acid (CAS 2809-21-4). The authors
increased water hardness, complexed the test substance with CaCl2 and
additionally performed a standard test which achieved 96 h EC50 values
of 8.8, 3.5 and 12 mg/l respectively based on cell numbers. While the
results are contrasting, the test does not reflect the true toxicity of
the test substance since essential nutrients such as Co and Fe will,
according to the ligand binding model and stability constants, continue
to be preferentially bound and thus not be bioavailable to the algae. In
the same manner results of a test carried out by HLS (2001) with
elevated nutrient levels (x25 times) to counterbalance nutrient
complexation by DTPMP-xNa (CAS 22042-96-2), will not be representative
of inherent toxicity since the amounts of essential nutrients added will
not be enough to counteract the phosphonates’ Fe and Co preferential
complexation and as a result the nutrients will remain unavailable,
inhibiting cell multiplication.
In addition SRI International (1984) tested the effects of EDTMP
acid with a diatom and two species of cyanobacteria while increasing the
nutrients in the test medium (x0.5 to x3 standard nutrient
concentrations) to counteract the complexing effects of phosphonates.
The general trend in the results supports that it is nutrient
complexation that is the cause of the effects seen in the studies.The
available evidence suggests that toxic effects observed in the tests are
a consequence of complexation of essential nutrients and not of true
toxicity. A study designed to ensure adequate levels of bioavailable
nutrients with either of the phosphonates would result in the test
substance being a phosphonates-Fe complex. Under conditions where iron
is readily available to counteract the effects of nutrient complexation
it is unlikely that the substance would have a negative effect on algal
growth (Girling et al. 2010). The nutrient complexing behaviour
of phosphonate substances therefore renders testing to determine their
intrinsic toxicity to algae impractical.
Prolonged (14-day) studies show a decrease in toxicity with time. For
example SRI International (1981) reports a 96 h ErC50 value
of 0.42 and a 14 d ErC50 value of 27 mg/l when
testing EDTMP acid with Selenastrum capricornutum (new name: Pseudokirchneriella
subcapitata) under standard conditions. This mitigation of effects
adds to the evidence that it is not inherent toxicity that is causing
the observed effects. This is thought to be attributable to the release
of phosphorous by the gradual photodegradation of the phosphonic
The interpretation of these data is also consistent with findings
presented in the risk assessment being carried out for the chelating
agent EDTA (CAS 60-00-4, Risk Assessment 2004), which is actually a
weaker complexing agent than BHMT. It has been demonstrated that for
EDTA it is not the absolute concentration, but rather the ratio of the
EDTA concentration to that of the metal cations that is crucial to
determining algal growth under the conditions of a toxicity test (EC,
The ability of iron to catalyse photodegradation of phosphonates
means that the interpretation of all algal growth data is somewhat
uncertain; this applies to the complexing agents discussed above
including EDTA. However, limitation of micronutrient availability is
considered to be a sufficiently generic phenomenon to explain effects
observed in toxicity tests with substances that have the capacity to
chelate cationic metals (Girling et al. 2010).
Available data on effects to algae and aquatic plants have been
reviewed and discussed in the peer-reviewed and published SIAR (please
refer to Section 4.1.3 of the SIAR). The conclusion(s) or critical
result(s) from the SIAR are as follows:
A total of nine results from tests with three freshwater genera were
available for consideration - two results from short-term (96-hour)
tests and seven results from prolonged-term tests (14 to 18 days). None
of the tests satisfied the requirements for achieving a reliability
rating of 1 but two short-term and two prolonged-term tests were of an
acceptable standard for assessing the toxicity of the substance. A
reliable short-term (96-hour) test with Selenastrum capricornutum yielded
an EC50, based on growth rate, of 3.0 mg/L. The lowest
reliable NOEC determined in the prolonged tests was 13 mg/L (14-day),
although there is evidence that the cultures did not remain in
exponential growth during the phase of the test extending from 96 hours
to 14 days. A 14-day LOEC of 1-10 mg/L and a 21-day NOEC of 3 mg/L were
also determined in other tests, the reliability of which could not be
A detailed interpretation of the effects of nutrient complexation by,
and photolytic release of phosphorus from, phosphonic acids on algal
growth in toxicity studies is given in Annex V to the phosphonic
acid SIARs (2004). The principle conclusions of the review are
growth may be stimulated by the presence of supplementary phosphorous
released by the photolytic degradation of phosphonic acids.
growth may be inhibited by the complexation of micronutrients (trace
metals) by phosphonic acids. This inhibition is an algistatic rather
than algicidal effect. Under the standard test conditions used for most
studies, the trace metals will be fully and strongly bound to the HEDP,
with the strong possibility that their bioavailability will have been
These two phenomena can occur at different stages in the course of the
same algal test and at different exposure levels of the substance.
The ability of iron to catalyse photodegradation of
phosphonates means that the interpretation of algal growth data can
be somewhat uncertain; this applies to the complexing agents
discussed above including EDTA. However, limitation of micronutrient
availability is considered to be a sufficiently generic phenomenon
to explain effects observed in toxicity tests with substances that
have the capacity to chelate cationic metals.
Conclusions: Great care has to be
exercised in interpreting the results of the algal tests carried out
with phosphonic acids. The significant potential for nutrient
complexation by phosphonates and/or release of phosphorous from
degradation of phosphonates to respectively either inhibit or
stimulate algal growth makes definitive interpretation difficult.
However the available evidence suggests that toxic effects observed
in tests with structurally analogous substances are a consequence of
complexation of essential nutrients and not of true toxicity. These
effects do not obey a classic dose response and as such
extrapolation using an assessment factor is inappropriate. In
addition, similar effects would not be anticipated in natural
environmental waters. Therefore further algal toxicity studies are
Please see the attached position paper which further
discusses algal tests with phosphonate substances and presents arguments
against further algal testing.
is a general term used to describe a molecule that bonds to
a metal; in the present case the phosphonate 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.
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