Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

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:

key study

Study period:

11/93

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

; directive 67/548/EEC (draft 1992)

Deviations:

yes

Remarks:

- only one replicate per concentration level and controls (insead of two); - the algal medium is not explicitly described (most likely described in cited SOP)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

- only one replicate per concentration level and controls (insead of two); - the algal medium is not explicitly described (most likely described in cited SOP)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All test concentrations, the control and the lowest and the highest concentration level without algae added were analysed, directly after start of the test and after 72 hours incubation time.

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Scenedesmus subspicatus CHODAT
- Source: Pflanzenphysiologisches Institut der Universität Göttingen

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

NA

Test temperature:

23°C+/-2°C

pH:

over all concentrations and control (t=0h): 7.9-8.2
over all concentrations and control (t=72h): 7.9-10.3

Nominal and measured concentrations:

Nominal concentrations in mg/L.: 15.6; 31.3; 62.5; 125; 250; 500; 1000
Measured concentrations in mg/L (t=0h): 18.5; 33.0; 64.0; 127.5; 264.5; 494.5; 1112.0
Measured concentrations in mg/L (t=72h): 17; 33.5; 67.0; 137.0; 258.0; 513.5; 1049.5
Control in mg/L (t=0h): < 1.0
Control in mg/L (t=72h): < 1.0
Measured concentration of 15.6 mg/L without algae added (t=0h): 17.0
Measured concentration of 15.6 mg/L without algae added (t=72h): 17.0
Measured concentration of 1000 mg/L without algae added (t=0h): 1030.0
Measured concentration of 1000 mg/L without algae added (t=72h): 1061.5

Details on test conditions:

- Light intensity and quality: 120 µE/m2*s
- Determination of cell density and biomass at: 24; 48; 72 hours
- Medium: 100 mL
- Counting procedure: Microcellcounter Sysmex F-300

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 33.3 - < 65.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 081 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: biomass area

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

140 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: biomass aerea

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17.8 mg/L

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: DUNNETT test

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

33.3 mg/L

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: DUNNETT test

Details on results:

The results relate to the mean of measured concentrations. The stability of the substance during the experiment was confirmed by analytical monitoring. The recovery rate was between 100.8% and 115.9 113.8%.

Reported statistics and error estimates:

DUNNETT test

According to the Guidance on information requirements and chemical safety assessment R.7b (ECHA, 2012) results based on the biomass should not be used for assessment since biomass results depend on the growth rate of the species as well as the test duration and other elements of the test design. Thus, the presented result was scored as not reliable.

Validity criteria fulfilled:

yes

Conclusions:

The results of the growth inhibition test of 2-phosphonobutane-1,2,4-tricarboxylic acid in algae is ErC50 (growth rate) > 1081 mg/L and a NOEC of 17.8 mg/L.

Executive summary:

In order to test acute toxicity to algae of 2-phosphonobutane-1,2,4-tricarboxylic acid, Desmodesmus subspicatus was exposed to the test solution of seven nominal concentrations of the test substance (15.6, 31.3, 62.5, 125, 250, 500 and 1000 mg/L) and blank control solution for a period of 72 h under static conditions. The study was conducted according to the OECD TG 201. The cell densities were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Analysis of all test concentrations at the beginning and at the end of the test yielded recovery rates between 100.8% and 113.8%, showing that the substance is stable during the experiment. The toxicity of growth inhibition showed a 72h-ErC50 of > 1081 mg/L; a 72h-NOEC of 17.8 mg/L and an 72h-ErC10 > 33.3 - < 65.5 mg/l was calculated. The results relate to measured concentrations as the stability of the substance during the experiment was confirmed.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

11/93

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Please find the Justification attached to Chapter 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

; directive 67/548/EEC (draft 1992)

Deviations:

yes

Remarks:

- only one replicate per concentration level and controls (insead of two); - the algal medium is not explicitly described (most likely described in cited SOP)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

- only one replicate per concentration level and controls (insead of two); - the algal medium is not explicitly described (most likely described in cited SOP)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All test concentrations, the control and the lowest and the highest concentration level without algae added were analysed, directly after start of the test and after 72 hours incubation time.

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Scenedesmus subspicatus CHODAT
- Source: Pflanzenphysiologisches Institut der Universität Göttingen

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

NA

Test temperature:

23°C+/-2°C

pH:

over all concentrations and control (t=0h): 7.9-8.2
over all concentrations and control (t=72h): 7.9-10.3

Nominal and measured concentrations:

Nominal concentrations in mg/L.: 15.6; 31.3; 62.5; 125; 250; 500; 1000
Measured concentrations in mg/L (t=0h): 18.5; 33.0; 64.0; 127.5; 264.5; 494.5; 1112.0
Measured concentrations in mg/L (t=72h): 17; 33.5; 67.0; 137.0; 258.0; 513.5; 1049.5
Control in mg/L (t=0h): < 1.0
Control in mg/L (t=72h): < 1.0
Measured concentration of 15.6 mg/L without algae added (t=0h): 17.0
Measured concentration of 15.6 mg/L without algae added (t=72h): 17.0
Measured concentration of 1000 mg/L without algae added (t=0h): 1030.0
Measured concentration of 1000 mg/L without algae added (t=72h): 1061.5

Details on test conditions:

- Light intensity and quality: 120 µE/m2*s
- Determination of cell density and biomass at: 24; 48; 72 hours
- Medium: 100 mL
- Counting procedure: Microcellcounter Sysmex F-300

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 33.3 - < 65.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 081 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: biomass area

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

140 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: biomass aerea

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17.8 mg/L

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: DUNNETT test

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

33.3 mg/L

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: DUNNETT test

Details on results:

The results relate to the mean of measured concentrations. The stability of the substance during the experiment was confirmed by analytical monitoring. The recovery rate was between 100.8% and 115.9 113.8%.

Reported statistics and error estimates:

DUNNETT test

According to the Guidance on information requirements and chemical safety assessment R.7b (ECHA, 2012) results based on the biomass should not be used for assessment since biomass results depend on the growth rate of the species as well as the test duration and other elements of the test design. Thus, the presented result was scored as not reliable.

Validity criteria fulfilled:

yes

Conclusions:

The results of the growth inhibition test of 2-phosphonobutane-1,2,4-tricarboxylic acid in algae is ErC50 (growth rate) > 1081 mg/L and a NOEC of 17.8 mg/L.

Executive summary:

As there is no data available for tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate a read-across approach with the corresponding parent acid 2-phosphonobutane-1,2,4-tricarboxylic acid is proposed.

In aqueous media, tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. Fate, behavior and the ecotoxicological properties of 2-phosphonobutane-1,2,4-tricarboxylic acid and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in environmental systems and have no relevant ecotoxic properties in low concentrations.

Therefore a read-across between tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphonobutane-1,2,4-tricarboxylic acid is justified.

In order to test acute toxicity to algae of 2-phosphonobutane-1,2,4-tricarboxylic acid, Desmodesmus subspicatus was exposed to the test solution of seven nominal concentrations of the test substance (15.6, 31.3, 62.5, 125, 250, 500 and 1000 mg/L) and blank control solution for a period of 72 h under static conditions. The study was conducted according to the OECD TG 201. The cell densities were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Analysis of all test concentrations at the beginning and at the end of the test yielded recovery rates between 100.8% and 113.8%, showing that the substance is stable during the experiment. The toxicity of growth inhibition showed a 72h-ErC50 of > 1081 mg/L; a 72h-NOEC of 17.8 mg/L and an 72h-ErC10 > 33.3 - < 65.5 mg/l was calculated. The results relate to measured concentrations as the stability of the substance during the experiment was confirmed.

Description of key information

As there is no data available for tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate, results of the parent acid, 2-phosphonobutane-1,2,4-tricarboxylic acid are taken into account for this endpoint.
The results of the growth inhibition test of 2-phosphonobutane-1,2,4-tricarboxylic acid in algae is an 72h-ErC50 of > 1081 mg/L , an 72h-ErC10 of > 33.3 - < 65.5 mg/l and a NOEC of 17.8 mg/L, based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

1 081 mg/L

EC10 or NOEC for freshwater algae:

33.3 mg/L

Additional information

As there is no data available for tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate a read-across approach with the corresponding parent acid 2-phosphonobutane-1,2,4-tricarboxylic acid is proposed.

In aqueous media, tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. Fate, behavior and the ecotoxicological properties of 2-phosphonobutane-1,2,4-tricarboxylic acid and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in environmental systems and have no relevant ecotoxic properties in low concentrations.

Therefore a read-across between tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphonobutane-1,2,4-tricarboxylic acid is justified.

In order to test acute toxicity to algae of 2-phosphonobutane-1,2,4-tricarboxylic acid, Desmodesmus subspicatus was exposed to the test solution of seven nominal concentrations of the test substance (15.6, 31.3, 62.5, 125, 250, 500 and 1000 mg/L) and blank control solution for a period of 72 h under static conditions. The study was conducted according to the OECD TG 201. The cell densities were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Analysis of all test concentrations at the beginning and at the end of the test yielded recovery rates between 100.8% and 113.8%, showing that the substance is stable during the experiment. The toxicity of growth inhibition showed a 72h-ErC50 of > 1081 mg/L; a 72h-NOEC of 17.8 mg/L and an 72h-ErC10 > 33.3 - < 65.5 mg/l was calculated. The results relate to measured concentrations as the stability of the substance during the experiment was confirmed. According to the Guidance on information requirements and chemical safety assessment R.7b (ECHA, 2012) results based on the biomass should not be used for assessment since biomass results depend on the growth rate of the species as well as the test duration and other elements of the test design. Thus, the presented result was scored as not reliable.

As recommended in REACH guidance document R.10 (ECHA, 2008), an EC10 for a long-term test which is obtained using an appropriate statistical method (usually regression analysis) will be used preferentially. The NOEC is defined as “the highest concentration tested at which the substance is observed to have no statistically significant effect (p< 0.05) when compared with the control, within a stated exposure period” (OECD TG 211, 1998) or the test concentration immediately below the LOEC, which when compared with the control has no statistically significant effect (p< 0.05) within a stated period (OECD TG 211, 1998). There has to be a concentration-effect relationship. In the past, the NOEC was mainly derived on the basis of ANOVA (analysis of variance) and a subordinate test (e.g. Dunett's) or determined directly from the concentration-effect curve by consideration of the deviation of the control (e.g. 10%). In older investigations, it may be difficult to find out how the NOEC was generated unless test reports or raw data are available. There has been a recommendation within OECD in 1996 to phase out the use of the NOEC, in particular as it can correspond to large and potentially biologically important magnitudes of effect. The preconditions for the use of ANOVA have to be fulfilled (normal distribution, homogeneous variances). The advantage of regression method for the estimation of ECx is that information from the whole concentration-effect relationship is taken into account and that confidence intervals can be calculated. These methods result in an ECx, where x is a low effect percentile (e.g. 5-20%).

In conclusion, it is not entirely clear as to whether the EC10 or NOEC from the algal study should be used as the main result, in the context of PNEC derivation. In this case, due to the shallow dose-response relationship seen in the study with Desmodesmus subspicatus, it is considered appropriate to use ErC10 as the primary result of the study.