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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Description of key information

Effects on algae are excluded from deriving a hazard assessment and consideration of PNEC.
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. The test organisms will be exposed to phosphonate-metal complexes.The effects seen in the studies will be a result of nutrient complexation rather than a reflection of the true toxicity of the test substance.

Key value for chemical safety assessment

Additional information

This section builds upon the categories “Phosphonic Acid Compounds Group 1, 2 and 3” which were reviewed and discussed in the peer-reviewed SIAR (finalised August 2005 following review and agreement at SIAM 18 in 2004; but the SIARs have not yet been published by OECD/UNEP). The information from the 2004 SIARs is utilised extensively in this position paper though the data set has been extended with data available on other phosphonates.

Available data on algal inhibition and the nutrient complexing properties of these substances have been reviewed and discussed in the peer-reviewed published SIAR (please refer to Annex V). The discussions and conclusion(s) from the SIAR are as follows:


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[1]. 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 (Girlinget 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 (Girlinget al. 2010). However, there is no evidence of severe toxicity from metal complexes of the ligands (Girlinget 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 (Girlinget al. 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 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 (Girlinget al. 2010). 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 (Girlinget 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 complexation strengths of phosphonates part of the Super category are in Table 7.4. They show that phosphonates are strong complexing agents, with stability constant values ranging from 5 to 24 (log10values)..

Table 7.4. Stability constants of phosphonates.


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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 EC50varying 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). They increased water hardness, binded the test substance with CaCl2and additionally performed a standard test which achieved 96 h EC50values 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 thenutrients 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 (SIAR 2005). 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 (Girlinget 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:Pseudokirchnerella 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 ATMP-N-oxide. 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, 2003).

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).

The principal and somewhat contrasting conclusions of the review are that:  

1)     Algal growth may be stimulated by the presence of supplementary phosphorous released by the photolytic degradation of phosphonic acids.

2)     Algal 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 EDTMP, with the strong possibility that their bioavailability will have been reduced considerably.

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. Therefore further algal toxicity studies are not recommended.

[1]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.