[nitrilotris(methylene)]trisphosphonic acid, ammonium salt

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

pH decreased significantly in the higher test concentrations

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Similar to OECD 201 but some replicates had additional CaCO3 added to the media

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA/600/4-89/001

Version / remarks:

Similar to the EPA guideline, but some replicates had additional CaCO3 added to the media

GLP compliance:

yes

Analytical monitoring:

no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: not reported
- Strain: not reported
- Source (laboratory, culture collection): not reported
- Age of inoculum (at test initiation): not reported
- Method of cultivation:not reported

ACCLIMATION
- Acclimation period:not reported
- Culturing media and conditions (same as test or not): not reported
- Any deformed or abnormal cells observed:not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 +/- 2 degrees C

pH:

Control 7.89, 0.1mg/L = 7.46, 5.0 mg/L = 7.78, 50 mg/L = 3.69, 100 mg/L = 3.16

Nominal and measured concentrations:

Nominal: control, 0.1, 5.0, 50.0 100.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 'flasks' no other details
- Type (delete if not applicable): open / closed not reported
- Material, size, headspace, fill volume: not reported
- Aeration: incubated on a mechanical shake table
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate):n/a
- Initial cells density: 10,000 cells/mL
- Control end cells density: not reported
- No. of organisms per vessel: not reported
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: yes - algal assay media (regular AAM)
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not reported
- Total organic carbon:not reported
- Particulate matter:not reported
- Metals:not reported
- Pesticides:not reported
- Chlorine:not reported
- Alkalinity:not reported
- Ca/mg ratio: not reported
- Conductivity:not reported
- Culture medium different from test medium:not reported
- Intervals of water quality measurement: beginning and end of test

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: not reported
- Photoperiod: continuous illumination
- Light intensity and quality: 400 +/- 60 ft-c
- Salinity (for marine algae): n/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

12.39 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active acid

Basis for effect:

biomass

Remarks on result:

other: 10.38-14.79 95% C.I.

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.

Chlorophyll a concentrations (ug/l) in Standard test medium at the end of  the test:

Test concentration (mg/l) Test 1  Test 2  Test 3
0 (Control)                  0.48          0.46          0.75        
0.1                           0.49          0.45          0.75
1.0                           0.62          0.53          0.82
5.0                           0.43          0.42          0.78
10                            0.35          0.46          0.62
50                           0.0009   0.0015   0.0022
100                           0.0002   0.0008   0.0012

Chlorophyll a concentrations (ug/l) in Hard medium at the end of the test:

Test concentration (mg/l)       Test 1        Test 2        
0 (Control)                         0.58        0.70                        
0.1                                 0.57        0.74                
1.0                                 0.53        0.75                
5.0                                 0.56        0.80                
10                                0.54        0.70                
50                                0.38        0.22        
100                                0.090        0.14        

EC50s were (1) 12.39 mg/l, (2) 19.14 mg/l and (3) 14.89 mg/l in tests  carried out in standard test media (<25 mg/l as CaCO3). 

EC50s determined in tests carried out in hard water (180 mg/l as CaCO3) were (1) 62.81 mg/l and (2) 33.88 mg/l.

Validity criteria fulfilled:

not specified

Remarks:

Not enough reported information in the report to compare against modern test guidelines (OECD 201) for each criterion.

Conclusions:

96 h EC50 value of 12.39 mg active acid/L have been determined for the effects of the test substance on the biomass of the freshwater alga, Pseudokirchneriella subcapitata.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 - 21 September 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Version / remarks:

Test was conducted according to standard operating procedure, BA225 version 02, which is based on ISO 10253

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: not reported
- Eluate:not reported
- Differential loading: not reported
- Controls: culture medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
- Other relevant information:

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: not reported
- Strain: CCAP 1077/1C
- Source (laboratory, culture collection): laboratory culture maintained under axenic conditions
- Age of inoculum (at test initiation): Not reported
- Method of cultivation: not reported

ACCLIMATION
- Acclimation period: not reported
- Culturing media and conditions (same as test or not):not reported
- Any deformed or abnormal cells observed:not reported

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

20 +/- 1 degree Celsius

pH:

not reported

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

Nominal concentrations were  2.1, 5.2, 13, 32, 80, 200 and 500 mg/l (as test substance).

Details on test conditions:

TEST SYSTEM
- Test vessel: borosilicate glass conical flasks of 250ml capacity
- Type (delete if not applicable): closed with polyurethane foam bungs
- Material, size, headspace, fill volume: 100 ml
- Aeration: continuous orbital shaking
- Type of flow-through (e.g. peristaltic or proportional diluter): N/a
- Renewal rate of test solution (frequency/flow rate):N/a
- Initial cells density: not reported
- Control end cells density: not reported
- No. of organisms per vessel: not reported
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/a

GROWTH MEDIUM
- Standard medium used: not reported
- Detailed composition if non-standard medium was used:not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:not reported
- Total organic carbon:not reported
- Particulate matter:not reported
- Metals:not reported
- Pesticides:not reported
- Chlorine:not reported
- Alkalinity:not reported
- Ca/mg ratio:not reported
- Conductivity:not reported
- Culture medium different from test medium: not reported but 'laboratory cultures maintained under axenic conditions'
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH:not reported
- Photoperiod: continuous light
- Light intensity and quality: cool white illlumination
- Salinity (for marine algae): not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell particle density was measured, no other information
- Chlorophyll measurement: not reported
- Other: Cell particle density measured at 24, 48 and 72 hours

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: not reported

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active acid

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active acid

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

80 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active acid

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not reported
- Unusual cell shape:not reported
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels:not reported
- Aggregation of algal cells: not reported
- Other: not reported
- Any stimulation of growth found in any treatment: Yes, in the 2.1, 5.2, 13, 32 and 80 mg/L treatments
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium:not reported

Reported statistics and error estimates:

The relative growth rates (expressed as % of control) were transformed to probability scale and analyzed by linear regression, against log concentration. The median effective concentrations (for area and growth rate) and their 95% confidence limits were determined from these data.

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.

RESULTS (expressed as active acid, as calculated by the reviewer):  

Area under growth curve:

No observed effect (P=0.05) concentration      =1.1 mg/l
Lowest significant effect (P=0.05)               =2.6 mg/l
Median effective concentration, biomass (EbC50) =65 mg/l
95% confidence limits                               =35-160 mg/l

The areas under the growth curves were equal to or greater than the control value for nominal test concentrations up to
and including 40 mg/l (active acid). The NOEC and LOEC values associated with a reduction in the areas under the
growth curves were:

No observed effect (P=0.05) concentration      =40 mg/l
Lowest significant effect (P=0.05)             =100 mg/l

Growth rates:

No observed effect (P=0.05) concentration      =40 mg/l
Lowest significant effect (P=0.05)               =100 mg/l
Median effective concentration, biomass (ErC50)=80 mg/l
95% confidence limits                               =41-230 mg/l

Validity criteria fulfilled:

not specified

Remarks:

Not enough reported information in the report to compare against modern test guidelines (OECD 201) for each criterion.

Executive summary:

72 h ErC₅₀ and NOErC values of 80 and 40 mg/L, have been determined for the effects of the test substance on the growth rate of the marine diatom, Skeletonema costatum.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Annex 3 of the CSR and IUCLID Section 13 for justification of read-across within the ATMP category.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active acid

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

80 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active acid

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active acid

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

12.39 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active acid

Basis for effect:

biomass

Remarks on result:

other: 10.38 - 14.79 95% C.I.

Description of key information

There are no test data for ATMP-xNH₄, however data have been read-across from ATMP-H

96-hour EC₅₀ 12.39 mg active acid/L, based on biomass, Pseudokirchneriella subcapitata, read-across from ATMP-H (this value is considered unreliable, due to pH deviations in the higher concentrations).

72-hour EC₅₀ and NOEC 80 and 40 mg active acid/L respectively, based on growth rate, Skeletonema costatum, read-across from ATMP-H

Ammonia: 5-day EC₅₀ 2.0 mg NH₃-N/L,Chlorella vulgaris. 14 d LOEC 3.0 mg NH₃-N/L, mixed algal populations.

Key value for chemical safety assessment

EC50 for marine water algae:

80 mg/L

EC10 or NOEC for freshwater algae:

2 mg/L

EC10 or NOEC for marine water algae:

40 mg/L

Additional information

ATMP-xNH₄will dissociate when it is released into aqueous environments to yield ATMP and ammonia. No data is available for ATMP ammonium salts, therefore data are read-across from ATMP-H and ATMP-xNa.

Two weight of evidence studies are available for ATMP-H. The 96-hour EC₅₀ value from the study with freshwater alga Pseudokirchneriella subcapitata (Monsanto, 1992) has been assigned a reliability 3 due to deviations in pH in the higher concentrations. Furthermore, this study used a non-standard exposure duration and is based on biomass. The standard 72-hour EC₅₀ and NOEC based on growth rate are 80 and 40 mg active acid/L respectively, but from a marine study, using the marine diatom, Skeletonema costatum. It is expected that the issues that arise from nutrient complexation by phosphonates and pH deviations will be lessened in saltwater, as it is better buffered and therefore, the values are expected to be higher, which they are.

Four studies are available for the effects of ATMP-H on different species of algae. An 18-day NOEC of < 10 mg/L was reported for the effects of ATMP-H on the reduction of biomass on Selenastrum sp. (Monsanto, 1972). Two 14-day NOEC values of 7.4 and 100 mg/L were reported for the effects of ATMP-H on Pseudokirchneriella subcapitata (SRI International, 1980 and Schoberl & Huber, 1988, respectively). A 14-day NOEC of 100 mg/L was reported for the effect of ATMP-H on the cell number of Selenastrum sp (Henkel, 1984).

The effects of ATMP observed in these tests with algae are likely to be a consequence of nutrient availability limitations caused by complexation and not true toxicity (see the discussion below). In these studies, the exposure durations are longer than the standard durations used in the OECD 201 test guideline (72 hours). Therefore, it is unlikely that exponential growth was maintained in these studies and therefore, these studies have been included as supporting studies. Also, due to the extended exposure durations in these studies, exponential growth of the algal cultures were unlikely to be maintained throughout the tests: 18 days (Monsanto, 1972) and 14 days (SRI International, 1980; Henkel, 1984 and Schoberi and Huber, 1988). The test results that are available cannot therefore be taken into account to assess the toxicity of ATMP. Two studies have been conducted with the sodium salts of ATMP. A 72-hour EC₅₀ value of >292 mg active acid/L and a NOEC value of 93 mg active acid/L have been determined for the effects of ATMP-xNa on the growth rate of the marine alga, Skeletonema costatum (TNO, 1996).

In a second study with Skeletonema costatum, 72-hour EC₅₀ and NOEC values of 465 and 282 mg/L respectively have been determined for the effects of ATMP-xNa on growth rate (AnalyCen, 2003).

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 true toxicity. It is therefore concluded that the nutrient complexing behaviour of phosphonate substances renders testing to determine their intrinsic toxicity to algae impractical. However, there is no evidence of severe toxicity from metal complexes of the ligands.

Ammonia: A 5-day EC₅₀ value of 2.0 NH₃-N/L has been determined for the effects of unionised ammonia on Chlorella vulgaris, reported in 'Environment Agency Proposed EQS for Water Framework Directive Annex VIII Substances: Ammonia (unionised) 2007' (EA, 2007). A 14-day LOEC value of 3.0 mg NH₃-N/L has been determined for the effects of unionised ammonia on mixed algal populations in the same report.

ATMP-xNH₄ will dissociate as it is released into the aquatic environment and it is therefore necessary to assess the hazard properties and derive PNECs for ATMP and ammonia separately.

For hazard classification, ATMP-xNH₄ has been assessed based on the available aquatic data for the whole substance.

The influence of the ammonium salt is discussed in IUCLID Section 6.0 Ecotoxicological Information and Section 7.0 of the CSR.

The acid, sodium, potassium and ammonium salts in the ATMP category are freely soluble in water. The ATMP anion can be considered fully dissociated from its sodium, potassium or ammonium cations when in dilute solution. Under any given conditions, the degree of ionisation of the ATMP 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 ATMP-H, ATMP.4Na, ATMP.7K or another salt of ATMP.

 

Therefore, when a salt of ATMP is introduced into test media or the environment, the following is present (separately):

1. ATMP is present as ATMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether ATMP (3-5K) salt, ATMP (3-5Na) salt, ATMP-H (acid form), or another salt was used for dosing.
2. Disassociated potassium, sodium or ammonium cations. The amount of potassium or sodium present depends on which salt was dosed.
3. It should also be noted that divalent and trivalent cations would preferentially replace the sodium or potassium ions. These would include calcium (Ca²⁺), magnesium (Mg²⁺) and iron (Fe³⁺). These cations are more strongly bound by ATMP than potassium, sodium and ammonium. This could result in ATMP-dication (e.g. ATMP-Ca, ATMP-Mg) and ATMP-trication (e.g. ATMP-Fe) complexes being present in solution.

In this context, for the purpose of this assessment, read-across of data within the ATMP 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^[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 2005). 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 “Modification of the growth media may be necessary for certain purposes, e.g. when testing metals and chelating agents or testing at different pH values.”  (OECD 201, 2011).

 

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, 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., 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.

Type	CAS Number	Ca	Cd	Co	Cu	Hg	Mg	Ni	Pb	Zn	Fe
ATMP	6419-19-8	7.6	12.7	18.4	17	21.7	6.7	15.5	16.4	14.1	approximately 25^a
ATMP-N-oxide-H	15834-10-3	5.69	No data	No data	No data	No data	8.29	No data	No data	No data	approximately 25^a
BHMT	34690-00-1	6.1^b	12^b	18^b	20^b	22^b	6.3^b	20^b	13^b	19^b	approximately 25^a
DTPMP-H	15827-60-8	6.7	9.7	17.3	19.5	22.6	6.6	19	8.6	19.1	approximately 25^a
HEDP	2809-21-4	6.8	15.8	17.3	18.7	16.9	6.2	15.8	No data	16.7	approximately 25^a
HMDTMP-H	23605-74-5	5.4	13.3	18.9	19.8	21.9	6	20.6	17	19.5	approximately 25^a
EDTMP-H	1429-50-1	9.6	21.4	15.8	24.3	31.8	10	20.2	23	21.1	approximately 25^a

Notes:

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 (Girlinget al. 2018).

 

All the algal toxicity studies available for phosphonates that have used standard and non-standard test conditions are presented in Girlinget al. (2018). 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 (96-hour 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-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 ATMP. 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 withSelenastrum capricornutumyielded 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 (2005). 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 phosphonate, 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 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.