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Toxicity to aquatic algae and cyanobacteria

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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 the result of nutrient complexation rather than a reflection of the true toxicity of the test substance.

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Additional information

In 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 substance's complexing properties of essential nutrients present in the test media.

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] (PFA 2009). In algal growth medium some metals form strongly-bound complexes and others form weakly-bound ones (PFA 2009). 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 (PFA 2009).

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 (PFA 2009, SIAR 2005). However, there is no evidence of severe toxicity from metal complexes of the ligands (PFA 2009).

In algal growth inhibition tests, complexation of essential trace nutrients (includingFe, Cu, Co, and Zn) by phosphonate 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 (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 (PFA 2009).

The SIAR (2005) 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 important divalent metal ions, cited as having been obtained from Monsanto internal reports (Owens, 1980). Values reported below are log10of the overall stability constant (Table 1 a and b).

The complexation constant for phosphonates with iron (III) has been estimated by TNO (1996a) to be around log K = 25 (PFA 2009).

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.

Table 1a Stability constants of phosphonatesfrom Lacour et al. (1999)

ATMP (X )

Ca

Cd

Cu

Ni

Zn

MX

7.6

12.2

17.4

11.3

16.3

MHX

16.6

19.4

23.8

19.6

22.5

HEDP (Y )

Ca

Cd

Cu

Ni

Zn

MY

6.7

8.7

12.0

8.6

10.3

MHY

14.7

16.3

17.4

15.6

16.6

 

 

 

Table 1b Stability constants of phosphonates from Gledhill and Feijtel (1992)

 

Ca

Cd

Co

Cu

Hg

Mg

Ni

Pb

Zn

ATMP

7.6

12.7

18.4

17.0

21.7

6.7

15.5

16.4

14.1

HEDP

6.8

15.8

17.3

18.7

16.9

6.2

15.8

Insol

16.7

DTPMP

6.7

9.7

17.3

19.5

22.6

6.6

19.0

8.6

19.1

 

Calculation 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 mean that key nutrients would still be complexed by the phosphonates 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 Co, Zn, Mn and Fe (PFA 2009). The resulting complexed nutrients will almost certainly not be bioavailable to aquatic plants and this can result in inhibited algal growth. Growth inhibition via this mechanism is a secondary effect and does not reflect the inherent toxicity of the test substance (PFA 2009).

 

A study designed to ensure adequate levels of bioavailable nutrients with either of the phosphonates would result in the actual substance tested 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 will have a negative effect on algal growth (PFA 2009). The nutrient complexing behaviour of phosphonate 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 (SIAR 2005).


[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.