2,5-dihydroxy-3,6-dimethyl-3,6-bis(dihydroxyphosphinyl)-1,4,2,5-dioxadiphosphorane-2,5-dioxide, hexasodium salt

Toxicity to aquatic algae and cyanobacteria

Description of key information

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. Any effects seen in the studies will be a result of nutrient complexation rather than a reflection of the true toxicity of the test substance.

Additional information

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] (Girling et al. 2010). In algal growth medium some metals form strongly-bound complexes and others form weakly-bound ones (Girling et al. 2010). 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 2005). 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 2005).

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 (SIAR 2005). 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 complexation constant for phosphonates with iron (III) has been estimated by TNO (1996a) to be around log K = 25 (Girling et al. 2010).

Calculations based on the known stability constants show that, even where the OECD-recommended approach to add additional calcium to the test media is used, that the key nutrients would still be complexed by the phosphonates in preference to complexation of calcium and magnesium. 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 Co, Zn, Mn and Fe (Girling et al. 2010). As a result the complexed nutrients will almost certainly not be bioavailable to aquatic plants and this can lead to inhibition of algal growth. Growth inhibition via this mechanism is a secondary effect and does not reflect the inherent toxicity of the test substance (Girling et al. 2010).

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 (Girling et al. 2010). The nutrient complexing behaviour of phosphonate substances therefore renders testing to determine their intrinsic toxicity to algae impractical.

A detailed interpretation of the effects of nutrient complexation, and photolytic release of phosphorus from, phosphonic acids on algal growth in toxicity studies is given in Annex V to the phosphonic acid SIARs (2005).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. The 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 ADPA Dimer Na Salt, 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.

Complexing agents, such as the phosphonates, inhibit algal growth as a consequence of their capacity to limit the bioavailability of trace metal micronutrients that are essential for growth. This has been illustrated by the following studies.

Hanstveit and Oldersma (1996) have conclusively demonstrated the importance of taking chelation/complexation into account in tests with another phosphonic acid, DTPMP. They have shown that DTPMP exhibits apparent toxicity (95-h ErC50 = 0.45 mg/l) to Selenastrum capricornutum in growth inhibition tests. These tests were carried out using standard OECD growth medium containing concentrations of Cu, Co and Zn that had been increased above the guideline concentration (Cu: up to 30 times, Co: up to 30x and Zn: up to 300x) in line with their predicted speciation. However, when the test medium was also supplemented with Fe at up to 300x the guideline concentration, no growth inhibition was observed at the highest test concentration of 10 mg/l. The explanation given for the absence of toxicity was that the addition of the Fe ensured that the free ion concentrations of all four of these essential nutrients were now in accordance with those specified in the standard OECD medium as being necessary for healthy algal growth. The experimental concept was for the iron to preferentially bind the DTPMP. The key role of Fe in determining the free concentration of the other elements was based on speciation calculations and an estimated value of the DTPMP-iron stability constant.

Similar findings have been reported by Schowanek et al (1996) from algal toxicity tests carried out with the unrelated chelating substance [S,S]-ethylene diamine disuccinate ([S,S]-EDDS). Chlorella vulgaris was tested according OECD 201, water hardness and trace metal concentrations were varied. In standard media with different water hardness (24-375 mg/l CaCO₃), addition of 1 mg/l [S,S]-EDDS reduced the growth rate by 53%, independent on the water hardness. Speciation calculations showed that in the standard medium [S,S]-EDDS is mainly associated with Zn, Cu, and Co. To test the hypothesis that the apparent toxicity was caused by nutrient deficiency, growth experiments in metal-enriched medium were performed. With increasing concentrations of Zn, Co, and Cu, the algal growth increased, reaching a maximum and then falling. The maximum growth was obtained with 1 mg/l (= 3.4 µM) [S,S]-EDDS, 0.62 µM Co, 0.051 µM Cu, and 2.9 µM Zn, where the levels of free Cu, Co and Zn were the same as in standard medium without the chelator. With lower [S,S]-EDDS concentrations, growth is decreased, mainly caused by Zn toxicity.

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 ADPA Dimer Na Salt and/or release of phosphorus from degradation of ADPA Dimer Na Salt 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.

References:

Girling, A.E. Fisk, P.R. Federici, G. (2010) REACH compliance of the phosphonates category: unsuitability of aquatic plant testing with complexing agents. Reference code: PFA.199.102.001. Owner Company Phosphonates Consortium.

SIAR Phosphonates (2005/6), SIDS Initial Assessment Report for SIAM 18, Phosphonic Acid Compounds Group 1, Amino tris(methylenephosphonic acid) and its sodium salts. Sponsor Country: United Kingdom, Shared partnership with: American Chemistry Council, Phosphonic Acids Compounds Panel