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Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions.
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
A test medium was designed with increased cobalt, copper, iron. and zinc concentrations to obtain constant free ion concentrations with increasing test substance concentrations.
GLP compliance:
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The algal growth medium was prepared from concentrated stock solutions in Milli-Q-filtered water, without Fe-citrate, Co-chloride. Cu-sulphate and Zn-sulphate, that are otherwise normal components of the medium. These latter components were added separately at varying concentrations.
The test medium was sterilised by micropore filtration, and contained 150 mg/l NaHC03 (not 50 mg/l as specified in the OECD Guideline in order to improve the buffer capacity of the medium.

The amount of the metals cobalt. copper, iron and zinc to be added to the medium in relation to the chosen DTPMP concentrations were calculated using the standard speciation programme; Titrator 2.2. The stability constants of the selected species were inferred from literature data. The stability constant (log) of Fe-DTPMP, however, was not found but was assumed to be approximately 25.

The following stock solutions of cobalt, copper and zinc were prepared:
- An amount of 15 mg CoCl2.6H20 was dissolved in 100 ml of ultra-pure Milli-Q water (pre-stock). Ten ml of this pre-stock was diluted up to 100 ml using ultra-pure Milli-Q water, yielding a stock solution containing a CoCl2.6H20
concentration of 15 mg/l.
- An amount of 15 mg CuS04.2H20 was dissolved in 100 ml of ultra-pure Milli-Q water (pre-stock). One ml of this pre-stock was diluted up to 100 ml using ultra-pure Milli-Q water, yielding a stock solution containing a CuS04.2H20 concentration of 0.15 mg/l.
- An amount of 63 mg ZnS04.7H20 was dissolved in 100 ml of ultra-pure Milli-Q water, yielding a stock solution containing a ZnS04.7H20 concentration of 630 mg/l.
- An amount of 800 mg Fe-citrate.3H20 was dissolved in 100 ml of ultra-pure Milli-Q water, yielding a stock solution containing a Fe-citrate.3H20 concentration of 8,000 mg/l.

The following solutions of the test substance DTPMP were prepared:
An amount of 110 mg DTPMP was dissolved in 100 ml of ultra-pure Milli-Q water, from which 600 ml dilutions in medium were prepared with addition of appropriate amounts of metal stock solutions, so as to yield a test substance concentration series of 0, 0.30, 1.0, 3.0 and 10 mg (a.i.)/l.

In one series the Co, Cu and Zn concentrations were increased, but not the Fe-concentration in order to illustrate the influence of iron on the test medium. In one series also the iron concentration was increased proportionally to the DTPMP concentration.

- Controls: Standard OECD medium without test substance

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): Pre-culture in exponential growth phase
Test type:
Water media type:
Limit test:
Total exposure duration:
95 h
Test temperature:
23 +/- 1°C
Start: pH 7.5
End of test:
Control without algae: 8.2 - 8.3
Control in standard medium with algae: 10.2
Nominal and measured concentrations:
Nominal: 0, 0.30, 1.0, 3.0 and 10 mg (a.i.)/l.
Details on test conditions:
Thirty 200 ml conical test flasks were covered with silicone sponge caps, autoclaved, coded and filled with a hundred ml of test substance solutions.
The complete test design is illustrated by the Table in "any other information on materials and methods including tables".
The standard OECD medium serves as the control. In one series the Co, Cu and Zn concentrations were increased, but not the Fe-concentration in order to illustrate the influence of iron on the test medium. In one series also the iron concentration was increased proportionally to the DTPMP concentration.
A suspension of exponentially growing algae was prepared by dilution of a pre-culture with algal growth medium. Addition of 1.0 ml of this algal suspension to 100 ml of the test media in the test flasks containing the test substance yielded a mean inoculum cell density, measured in the control cultures at the start of the test of 1.05 x 10^4 cells/ml.

The test was carried out in duplicate with two controls with standard OECD-medium containing algae only (i.e. without additional metals and DTPMP), and a single background series containing test substance without algae.

- Test vessel: Conical flask
- Type (delete if not applicable): covered with silicone sponge caps
- Material, size, headspace, fill volume: Glass, Size: 200 ml, Fill volume: 100 ml
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Static
- Initial cells density: 1.05 x 10^4 cells/ml.
- Control end cells density:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates): 2

- Standard medium used: no

- Source/preparation of dilution water: Milli-Q
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: The pH was measured at the start (medium without algae) and after 95 h in selected cultures.
All flasks were incubated at 23 °C and shaken (approximately 100 rpm) in an orbital shaker.

- Adjustment of pH: no
- Photoperiod: not reported
- Light intensity and quality: The light intensity radiated by the fluorescent lamps was within the standard range of 60-120 µmol.s-1.m-2.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] One sample was taken from each flask after 0, 23.5, 44.5, 70.5 and 95 h, and the number of algal cells per ml in the samples was analysed with the aid of a Coulter Counter model TAIL.
The morphology of the algae was examined visually with the aid of a microscope at the start and end of the test.

Reference substance (positive control):
95 h
Dose descriptor:
Effect conc.:
> 10 mg/L
Nominal / measured:
Conc. based on:
other: active acid
Basis for effect:
growth rate
95 h
Dose descriptor:
Effect conc.:
>= 10 mg/L
Nominal / measured:
Conc. based on:
other: active acid
Basis for effect:
growth rate
Reported statistics and error estimates:
The EC50 values and NOEC were estimated by visual comparison of the measured and calculated growth values of the treated algal suspensions with those of the controls.

The free ion concentration of Cu, Co, and Fe in the presence of varying concentrations of DTPMP were calculated. In addition the free ion concentration of DTPMP was calculated.

Table: Nominal concentrations of DTPMP added. and corresponding free ion concentrations in OECD test medium amended with Co, Cu, Ee and Zn

 Conditions  Nominal concentration DTPMP mg/l  Nominal concentrations DTPMP 10 log M  Calculated free ion concentration (10 log M)        
       DTPMP  Cu  Co  Zn  Fe
 Standard OECD medium  0  0  -24  -14.99  -11.54  -10.956  -18.55
 Amended OECD medium with standard Fe content  0.3  -6.28  -10.49  -19.24  -15.04  -15.27 -21.09 
   1.0  -5.76  -9.12  -20.12  -15.90  -16.16  -22.25
   3.0  -5.28  -8.59  -20.13  -15.91  -16.17  -22.99
   10  -4.76  -7.99  -20.26  -16.03  -16.29  -23.58
 Amended OECD medium 0.3   -6.28  -14.52 -15.22   -11.61  -11.26  -16.99
   1.0  -5.76  -14.47  -14.78  -11.12  -10.83  -16.49
   3.0  -5.28  -14.57  -14.16  -10.57  -10.22  -15.94
   10  -4.76  -14.48  -13.77  -10.11  -9.82  -15.47

The calculations demonstrated that addition of DTPMP to the medium only amended by a higher zinc concentration, resulted in a drastic reduction of the free ion concentrations of the four metals. Adjustment of the metals concentration further was not sufficient to maintain their free ion concentrations.

However, by adjusting the iron concentration proportionally to the concentration of DTPMP, the free metals concentrations were maintained at a level comparable to that of the standard OECD test medium.

It was therefore predicted that in the concentration series tested in medium with constant Fe concentrations a growth inhibition should be observed. In the other series, little or no effect of DTPMP addition on algal growth should be found.

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.

There were no effects on growth at the highest test concentration (10 mg/l)

Reliable 95 h ErC50 and NOErC values of >10 and ≥10 mg active acid/l has been determined for the effects of DTPMP-H towards the growth rate of the freshwater alga Pseudokirchneriella subcapitata (reported as Selenastrum capricornutum). Also, it is concluded that growth inhibition observed by DTPMP addition in standard OECD 201 test medium is not caused by its intrinsic toxicity, but by complexing of essential elements (most probably Cu and Fe) and the Fe addition in the test medium has a crucial effect on the free ion concentrations of the essential elements as long as its complex stability constant is higher than that of the other elements.

DTPMP can undergo photodegradation in water under laboratory conditions in solutions supplemented with metals, especially iron (III), decreasing the DTPMP concentration and releasing orthophosphate and other by-products. In this study, additional metal stock solutions were added to the algal test medium and the ionisation state of the iron (added as Fe-citrate.3H2O) is expected to be iron (III) based on the pH range of the algal media throughout the study (pH 7.5 – 10.2). The test vessels were held under fluorescent lamps with a light intensity between 60 and 120 µmol s-1 m-2 . While the test conditions and experimental set-up are not directly comparable, it cannot be ruled out that some photodegradation of DTPMP could have occurred during the course of this study. Nevertheless, the steps taken in this test appear appropriate and effective to counteract the indirect toxic effects to algae of mineral depletion, caused by complexation of essential metals by DTPMP.

Description of key information

95 h ErC50 and NOErC values of >10 and ≥10 mg active acid/l, Pseudokirchneriella subcapitata (reported as Selenastrum capricornutum), (TNO, 1996a, read-across from DTPMP-H). This test was selected as the key study because additional metal stock solutions were added to the test media to attempt to reduce the effects of nutrient complexation.

Due to the essential nutrients present in the test medium being complexed by the phosphonates, it is likely that the test organisms were exposed to phosphonate-metal complexes. The effects seen in the studies may 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

A total of four results from tests with two species (one freshwater species, Selenastrum capricornutum, and one marine species, Skeletonema costatum) are available for consideration on the effects of the DTPMP category to algae. All studies were of an acceptable standard for assessing the toxicity of the substance and its salts. The tests that were carried out over exposure periods ranging from 69 to 96 hours yielded EC50 values in the range 1.54 to 36 mg/L for both DTPMP and its salts when the results are expressed as active acid. Marine and freshwater species exhibited similar responses when test results are compared on the basis of active acid content of the test media. The NOECs in the tests ranged from 0.45 to 10 mg/L as active acid.

It is possible that some photodegradation of DTPMP could have occurred during the algal studies, particularly when supplementary iron (III) is used. The selection of the TNO, 1996a study as a key study was based on the attempts to counteract the secondary toxic effects of mineral depletion, caused by complexation of metals in the media, by adding increased concentrations of several metal ions. Despite the uncertainties applying, this study is the most likely of those available to be indicative of true toxicity.  For further information on the photodegradation of DTPMP in water, please refer to IUCLID Section 5.1 and Section 4.1.1 of the CSR.


The acid and salts in the DTPMP category are freely soluble in water and, therefore, the DTPMP anion is fully dissociated from its cations when in solution. Under any given conditions, the degree of ionisation of the DTPMP 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 DTPMP-H, DTPMP (1-3Na), DTPMP (5-7Na), DTPMP (4-8K), DTPMP (xNH4) or another salt of DTPMP.


Therefore, when a salt of DTPMP is present in test media or the environment, the following is present (separately):

1. DTPMP is present as DTPMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether DTPMP-H, DTPMP (1-3Na), DTPMP (5-7Na), DTPMP (4-8K), DTPMP (xNH4), or another salt was used for testing.

2. Disassociated ammonium, potassium or sodium cations. The amount of ammonium, potassium or sodium present depends on which salt was added.

3. Divalent and trivalent cations have much higher stability constants for binding with DTPMP than the sodium, potassium or ammonium ions so would preferentially replace them. These ions include calcium (Ca2+), magnesium (Mg2+) and iron (Fe3+). Therefore, the presence of these in the environment or in biological fluids or from dietary sources would result in the formation of DTPMP-dication (e.g. DTPMP-Ca, DTPMP-Mg) and DTPMP-trication (e.g. DTPMP-Fe) complexes in solution, irrespective of the starting substance/test material.

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[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 (Girling et al. 2018).


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. 2018, SIAR 2004). However, there is no evidence of severe toxicity from metal complexes of the ligands (Girling et al. 2018).


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 following table.


Table: Stability constants of phosphonates.


CAS Number






















approximately 25a




No data

No data

No data

No data


No data

No data

No data

approximately 25a












approximately 25a












approximately 25a










No data


approximately 25a












approximately 25a












approximately 25a



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. 2018).

All the algal toxicity studies available for phosphonates that have used standard and non-standard test conditions are presented in Girling et al. (2018). 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. 2018). 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 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 HEDP. 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. 2018).


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 assessed.


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:


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


·        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 HEDP, 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. 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 not recommended.

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.


[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.