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EC number: 267-956-0
CAS number: 67953-76-8
Table 1. Reinterpretation of
data according to OECD TG 201 by study reviewer
Nominal concentration (mg active acid/L)
Day 0 cell concentration (cells/ml)
Day 4 cell concentration (cells/ml)
96h Average specific growth rate
Result expressed as nominal concentration. Properties of the
test substance and evidence from other studies (where
concentrations were measured) indicate that nominal and
measured concentrations are likely to be in good agreement.
Mean cell concentrations (cells/ml) after 96 hours and 14 days, as
reported by study report
Nominal test concentration (mg/l)
96 hr EC50: 3 mg/l, 96 hr NOEC: 0.74 mg/l, as reported by study report.
Toxicity decreased with time.
At 14 days cultures exposed to 7.4 and 13.22 mg/l
concentrations showed increased cell growth compared to controls.
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.
96-hour ErC50 >132.22 mg active acid/L and NOErC
13.22 mg active acid/L, Pseudokirchneriella subcapitata, read-across
A 96-hour EbC50 value of 3 mg active
acid/L based on biomass and 14-day NOEC and LOEC values of 13 mg
active acid/L and 39 mg active acid/L, respectively, based on growth
rate were reported in the key study with HEDP-H (SRI International,
1980). However, the EbC50 value is based on
biomass and the control group did not demonstrate exponential growth
over the 14-day exposure period. Therefore, a 96-hour ErC50
value of >132.22 mg active acid/L and a NOErC
value of 13.22 mg active acid/L have been determined for the effects
of HEDP-H on the growth rate of Pseudokirchneriella subcapitata by
the study reviewer, in accordance with OECD TG 201 calculations.
These values have been selected as key for the endpoint as they
represent a standard exposure duration in which exponential growth
was maintained in the control group.
Several supporting algal studies of reliability rating 4, are
available with HEDP acid and its salts. Two results are available
for HEDP (2-3Na): a 14-day LOEC value of 30 mg/L was determined for
the effects on the cell number of Selenastrum sp. and a
48-day EC50 value of >960 mg/L was determined for the
effects on the cell number of Chlorella vulgaris (Henkel,
1984a and b). Three 18-day NOEC values of 10, 10 and <10 mg/L have
been determined for the effects of HEDP-H on the biomass of Chlorella
sp., Anabaena sp. and Selenastrum sp.,
respectively (Monsanto, 1972a, b and c).
A reliable study reported 96-hour EC50 values of
12, 2.5 and 8.8 mg active acid/L for the effects of HEDP-xNH4
on the cell number of P. subcapitata in: normal test media
conditions; test media with added calcium; and test media with added
calcium and magnesium conditions, respectively (Springborn, 1992).
NOEC values of 50, <6.2 and <6.2 mg active acid/L were reported for
the effects of HEDP-xNH4 on the cell number of P.
subcapitata under the same respective test media conditions
(Springborn, 1992). A LOEC value of 1 -10 mg/L was reported for the
effects of HEDP-H to P. subcapitata, however the study was
assigned reliability 4 due to a lack of documentation (Schoberl and
A supporting study is available that reports EC50
values of 7.23 mg/L, 14.96 mg/L and 0.07 mg/L for the effects of
HEDP-H on the biomass of P. subcapitata in test media and
hard water, however it was assigned a reliability rating of 3, based
on the reduction in pH, which decreased to 3.11 in the highest test
concentration (Monsanto, 1992).
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 (2-3Na). 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
The acid, sodium and potassium salts in the HEDP category are
freely soluble in water and, therefore, the HEDP anion is fully
dissociated from its sodium or potassium cations when in solution.
Under any given conditions, the degree of ionisation of the HEDP
species is determined by the pH of the solution. At a specific pH, the
degree of ionisation is the same regardless of whether the starting
material was HEDP-H, HEDP (1-2Na), HEDP (2-3Na), HEDP-4Na, HEDP-xK or
another salt of HEDP.
Therefore, when a salt of HEDP is introduced into test media or
the environment, the following is present (separately):
In this context, for the purpose of this assessment, read-across
of data within the HEDP Category is considered to be valid.
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.
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. 2018).
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-hour 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-hour ErC50
value of 0.42 and a 14-day 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 substances.
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 that:
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 (Girling et al,
2018) which further discusses algal tests with phosphonate
substances and presents arguments against further algal testing.
 'Ligand’ 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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