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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From2012-07-16 to 21 february 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with standard test guidelines and GLP conditions, with no deviations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
2012-11-30
Analytical monitoring:
yes
Details on sampling:
Verification of Test Concentrations:
Samples were taken from the control (replicates R1 – R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative
analysis. All samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at
approximately 20ºC for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Range-Finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg HPA/L for a period of 72 hours. All test item concentrations were corrected for a test item water content of 50%.

The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to
reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.

An amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg HPA/L stock solution from
which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg HPA/L. The pH of each of the stock solutions was
measured using a WTW pH 320 pH meter and adjusted to pH 7.5. An aliquot (450 mL) of each of the stock solutions was separately inoculated with
algal suspension (5.9 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg HPA/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter®
Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at
24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken
at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test
conditions. All samples were stored at approximately -20 °C prior to analysis.


Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 20, 40, 80 and 160 mg HPA/L.


Experimental Preparation
For the purpose of the definitive test, the test item was dissolved directly in culture medium. All test concentrations were corrected for a test item
water content of 50%.

An amount of test item (320 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 160 mg HPA/L stock solution from
which a series of dilutions was made to give further stock solutions of 80, 40, 20 and 10 mg HPA/L. The pH of each of the stock solutions was
measured using a WTW pH 320 pH meter and adjusted to pH 7.5. An aliquot (500 mL) of each of the stock solutions was separately inoculated with
algal suspension (9.5 mL) to give the required test concentrations of 10, 20, 40, 80 and 160 mg HPA/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.


Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test Species
The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from
the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under
constant aeration and constant illumination at 21 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 - 105 cells/mL.






Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 10, 20, 40 and 80 mg HPA/L, however misshapen cells were observed to be present in the test cultures at 160 mg HPA/L.
Hardness:
Not recorded.
Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test
pH:
The pH value of the control cultures (see Table 2) was observed to increase from pH 7.6 at 0 hours to pH 7.8 at 72 hours. The pH deviation in the
control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
Dissolved oxygen:
Not recorded.
Salinity:
not applicable
Nominal and measured concentrations:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 20, 40, 80 and 160 mg HPA/Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 87% to 92% of nominal and so the results are based on nominal HPA test concentrations only.
Details on test conditions:
Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture (see composition in Appendix 3 in the Overall remarks, attachement field).

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group. The control group was maintained under identical conditions but not exposed to the test item. A positive control (Harlan Study Number: 41300097) used potassium dichromate as the reference item. Details of the positive control are given in the attached Appendix 2. The positive control was conducted between 15 January 2013 and 18 January 2013.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 2.64 x 105 cells per mL. Inoculation of
500 mL of test medium with 9.5 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous
illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


Physico-Chemical Measurements
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.


Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
98 other: mg HPA/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 160 other: mg HPA/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
40 other: mg HPA/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
80 other: mg HPA/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
140 other: mg HPA/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
40 other: mg HPA/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
80 other: mg HPA/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the
range-finding test are given in Table 1.

The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 mg HPA/L. However, growth was observed to be reduced at 100 mg/L.

Based on this information test concentrations of 10, 20, 40, 80 and 160 mg HPA/L were selected for the definitive test.

Chemical analysis of the 10 and 100 mg HPA/L test preparations at 0 and 72 hours (see attached Appendix 4) showed near nominal concentrations were obtained with the exception of the 10 mg HPA/L test preparation at 0 hours where a measured concentration of 67% of nominal was obtained. This result was considered to be erroneous given that the corresponding sample at 72 hours showed a measured concentration of 99% of nominal
was obtained. As such it was considered that the test item was stable over the test period.


Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

The mean cell densities versus time for the definitive test are presented in the attached Figure 1. Percentage inhibition values are plotted against test
concentration in the attached Figure 2 and Figure 3.


Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 117 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.97 x 10E+3 cells per mL
Mean cell density of control at 72 hours : 6.99 x 10E+5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 14% and hence satisfied the validation
criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the
validation criterion given in the OECD Guideline which states that this must not exceed 7%.


Growth Data
From the data given in Table 2 and Table 4 it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h) : 98 mg HPA/L
ErC20 (0 - 72 h) : >160 mg HPA/L
ErC50 (0 - 72 h) : >160 mg HPA/L

where ErCx is the test concentration that reduced growth rate by x%.


Inhibition of Yield
EyC10 (0 - 72 h) : 32 mg HPA/L
EyC20 (0 - 72 h) : 55 mg HPA/L
EyC50 (0 - 72 h) : 140 mg HPA/L

Where:

EyCx is the test concentration that reduced yield by x%.


Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test
cultures were observed to be green dispersions.


Physico-Chemical Measurements
The pH values of the control and each test preparation are given in Table 2. Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH of the stock solutions was adjusted to pH 7.5 prior to inoculation with algal cells.

The pH value of the control cultures (see Table 2) was observed to increase from pH 7.6 at 0 hours to pH 7.8 at 72 hours. The pH deviation in the
control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.


Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 87% to 92% of nominal and so the results are based on nominal HPA test concentrations only (see Table 5 in the Overall remarks, attachements field).
Results with reference substance (positive control):
Positive Control
A positive control (Harlan Study Number: 41300097) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25,
0.50 and 1.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:

ErC50 (0 – 72 h) : 0.89 mg/L*
EyC50 (0 – 72 h) : 0.36 mg/L; 95% confidence limits 0.33 – 0.41 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

* It was not possible to calculate 95% confidence limits for the EyC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments
with a control (Dunnett, 1955). There were no statistically significant decreases in growth rate between the control, 10, 20 and 40 mg HPA/L test
concentrations (P ≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect
Concentration" (NOEC) based on growth rate was 40 mg HPA/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on
growth rate was 80 mg HPA/L.

Statistical analysis of the yield data was carried out. There were no statistically significant decreases in yield between the control, 10, 20 and 40 mg HPA/L test concentrations (P ≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed
Effect Concentration" (NOEC) based on yield was 40 mg HPA/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 80 mg HPA/L.

Table1     Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration

(mg HPA/L)

Cell Densities* (cells per mL)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

5.57E+03

2.55E+05

-

-

 

R2

6.01E+03

3.43E+05

 

Mean

5.79E+03

2.99E+05

0.10

R1

7.66E+03

2.79E+05

9

17

 

R2

5.84E+03

2.20E+05

 

Mean

6.75E+03

2.49E+05

1.0

R1

6.63E+03

3.49E+05

5

[12]

 

R2

9.01E+03

3.20E+05

 

Mean

7.82E+03

3.34E+05

10

R1

6.72E+03

2.43E+05

9

8

 

R2

7.80E+03

3.08E+05

 

Mean

7.26E+03

2.75E+05

100

R1

7.42E+03

1.97E+05

20

44

 

R2

6.98E+03

1.46E+05

 

Mean

7.20E+03

1.71E+05

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks. In accordance with the OECD test guideline inhibition of growth was calculated based on measured 0-Hour cell densities whilst inhibition of yield was calculated based on a nominal cell density of 5.00 x 103cells/mL

R1and R2= Replicates 1 and 2          

[Increase in growth compared to controls]

Table2     Cell Densities and pH Values in the Definitive Test

Nominal Concentration

(mg HPA/L)

pH

Cell Densities* (cells per mL)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

7.6

5.44E+03

2.68E+04

9.23E+04

5.44E+05

7.8

 

R2

5.71E+03

2.83E+04

1.28E+05

7.73E+05

 

R3

6.27E+03

2.92E+04

9.64E+04

6.46E+05

 

R4

5.84E+03

2.92E+04

1.49E+05

7.82E+05

 

R5

6.69E+03

2.73E+04

9.88E+04

6.94E+05

 

R6

5.87E+03

2.73E+04

1.21E+05

7.56E+05

 

Mean

5.97E+03

2.80E+04

1.14E+05

6.99E+05

10

R1

7.5

8.00E+03

2.82E+04

1.29E+05

1.09E+06

7.7

 

R2

6.50E+03

2.88E+04

1.55E+05

1.17E+06

 

R3

7.17E+03

2.86E+04

1.12E+05

7.29E+05

 

Mean

7.22E+03

2.85E+04

1.32E+05

9.98E+05

20

R1

7.5

7.36E+03

2.62E+04

1.09E+05

7.59E+05

7.7

 

R2

7.57E+03

2.60E+04

1.08E+05

7.90E+05

 

R3

8.59E+03

2.87E+04

1.34E+05

7.22E+05

 

Mean

7.84E+03

2.69E+04

1.17E+05

7.57E+05

40

R1

7.5

8.94E+03

2.19E+04

7.19E+04

5.56E+05

7.7

 

R2

8.03E+03

2.63E+04

9.07E+04

6.66E+05

 

R3

8.38E+03

2.42E+04

1.00E+05

4.96E+05

 

Mean

8.45E+03

2.41E+04

8.76E+04

5.73E+05

80

R1

7.5

8.74E+03

1.78E+04

6.16E+04

3.19E+05

7.7

 

R2

1.23E+04

1.91E+04

5.55E+04

3.27E+05

 

R3

9.49E+03

2.09E+04

6.89E+04

3.30E+05

 

Mean

1.02E+04

1.93E+04

6.20E+04

3.25E+05

160

R1

7.5

6.88E+03

2.44E+04

9.31E+04

3.77E+05

7.7

 

R2

8.66E+03

2.55E+04

9.32E+04

4.52E+05

 

R3

6.77E+03

2.42E+04

1.09E+05

4.53E+05

 

Mean

7.43E+03

2.47E+04

9.84E+04

4.28E+05

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks. In accordance with the OECD test guideline inhibition of growth was calculated based on measured 0-Hour cell densities whilst inhibition of yield was calculated based on a nominal cell density of 5.00 x 103cells/mL

R1- R6= Replicates 1 to 6

Table 3     Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/mL/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.070

0.052

0.074

 

R2

0.072

0.063

0.075

 

R3

0.074

0.050

0.079

 

R4

0.074

0.068

0.069

 

R5

0.071

0.054

0.081

 

R6

0.071

0.062

0.076

 

Mean

0.072

0.058

0.076

R1- R6= Replicates 1 to 6

Table4     Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration
(mg HPA/L)

Growth Rate

(cells/mL/hour)

Yield

(cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.065

 

5.38E+05

 

 

R2

0.070

 

7.67E+05

 

 

R3

0.068

 

6.40E+05

 

 

R4

0.070

-

7.76E+05

-

 

R5

0.069

 

6.87E+05

 

 

R6

0.070

 

7.50E+05

 

 

Mean

0.069

 

6.93E+05

 

 

SD

0.002

 

9.22E+04

 

10

R1

0.075

[9]

1.08E+06

 

 

R2

0.076

[10]

1.17E+06

 

 

R3

0.069

0

7.22E+05

 

 

Mean

0.073

[6]

9.90E+05

[43]

 

SD

0.004

 

2.36E+05

 

20

R1

0.070

[1]

7.52E+05

 

 

R2

0.070

[1]

7.82E+05

 

 

R3

0.069

0

7.14E+05

 

 

Mean

0.070

[1]

7.49E+05

[8]

 

SD

0.001

 

3.43E+04

 

40

R1

0.065

6

5.47E+05

 

 

R2

0.068

1

6.58E+05

 

 

R3

0.064

7

4.88E+05

 

 

Mean

0.066

5

5.64E+05

19

 

SD

0.002

 

8.65E+04

 

80

R1

0.058

16

3.10E+05

 

 

R2

0.058

16

3.14E+05

 

 

R3

0.058

16

3.20E+05

 

 

Mean

0.058

16

3.15E+05

55

 

SD

0.000

 

5.13E+03

 

160

R1

0.060

13

3.70E+05

 

 

R2

0.063

9

4.44E+05

 

 

R3

0.063

9

4.47E+05

 

 

Mean

0.062

10

4.20E+05

39

 

SD

0.002

 

4.32E+04

 

*In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated

R1-R6= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Analytical Tables

Table1     Preparation of Test Samples

Nominal concentration of test item [mg HPA/L]

Sample Volume

[mL]

Final Volume

[mL]

Sample preparation factor

F

Final Nominal Concentration of test item

[mg HPA/L]

Control

100

100

1

-

10

100

100

1

10

20

10

20

2

10

40

5

20

4

10

80

5

25

8.3

6.9

160

5

50

16.7

9.6

  -   =  not applicable  

 

Table2     Linearity Data

 

Concentration of Test Item

 

CPS

[mg HPA/L]

[counts]

0

1.206 x 103

0.548

4.368 x 103

1.095

7.391 x 103

5.475

3.079 x 104

10.950

5.922 x 104

15.630

8.493 x 104

16.425

8.677 x 104

21.900

1.116 x 105

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results
corrected for the water content of the test item:
Response Variable EC50 (mg HPA/L) No Observed Effect Concentration Lowest Observed Effect
(NOEC) (mg HPA/L) Concentration (LOEC) (mg HPA/L)
Growth Rate >160 40 80
Yield 140 40 80


Executive summary:

A study was perform to assess the effect of the test item on the growth of the green alga Desmodesmus subspicatus. The method followed was designed to be compatible with the OECD Guidelines No 201 and EU C.3 of Commission Regulation (EC) No 761/2009.

 Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test item at concentrations of 10, 20, 40, 80 and 160 mg HPA (Phosphinic acid)/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 The test item is a solution of phosphinic acid containing 50% water. All test concentrations were corrected for the test item water content of 50%. Due to the acidic nature of the test item, the pH of the test preparations was adjusted to pH 7.5 prior to inoculation with algal cells.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a counter.

Exposure of Desmodesmus subspicatus to the test item gave the following results corrected for the water content of the test item:

 

Response Variable

  

EC10

(mg HPA/L) 

EC50

(mg HPA/L)

No Observed Effect Concentration (NOEC)

(mg HPA/L)

Lowest Observed Effect Concentration (LOEC)

(mg HPA/L)

Growth Rate

  98

>160

40

80

Yield

  32

140

40

80

 

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 87% to 92% of nominal and so the results are based on nominal HPA test concentrations only.

 

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Justification of the read across:
In the current assessment, data from sodium phosphinate were used to evaluate the phosphinic acid properties. Phosphinic acid is commercially prepared as the result of pH adjustement of the sodium phosphinate salt. The main assumption is that sodium is not significant in respect of all the properties under consideration which are expected to be related to the phosphinate anion.
In dilute aqueous conditions of environmental pH (5-9) the salt will behave no differently to the parent acid, at identical concentration of the particular speciated form present and will be fully dissociated. For the aquatic hazard assessment, some tests were performed on monohydrate salt and the results on measured active compounds (phosphinate) are expressed on phosphinic acid to be used for the purpose of risk assessment and classification and labelling. Therefore the read across approach is applied in the present dossier.

Please also refer to the Read across Justification document provided in Section 13
Reason / purpose for cross-reference:
read-across source
Vehicle:
no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes (see table 1 in remarks on results section)
- Observation of abnormalities (for algal test): no differences in shape and size of the algal cells was observed
- Any stimulation of growth found in any treatment: no (see table 2 in remarks on results section)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period

No more data available
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
February 1995
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is claimed to follows the OECD Guideline 201, but there is no data regarding GLP compliance, and stability of the substance through the test. Furthermore 3 validity criteria were not respected (pH in control, initial biomass and increase cell density) and there is no information on raw biomass data specific growth criteria. In absence of more detailed information on raw biomass data, analytical monitoring and pH adjustment, we cannot excluded that validity criteria deviations (pH variation, or initial biomass and cell density) may have affected the results of the test. As a consequence the test is considered as invalid for the assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
inoculum of 2.47x10E04 cells/mL instead of 2-5x10E03 cells/mL
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: direct dispersion in culture medium
- Controls: yes in culture medium without test substance
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 276/20
no more data

ACCLIMATION
no data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
24°C
pH:
the pH of the culture medium after equilibration with air is approximately 8
pH values at 0 and 72 hours for all test concentrations were between 8 and 10.3 (for details see table 2 in freetext)
Dissolved oxygen:
no data but cultures were under continuous stirring (orbital shaker) during the test
Nominal and measured concentrations:
nominal concentrations : 0.625; 1.25 ; 2.5; 5.0 and 10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type : closed, all flasks have been loosely stoppered to reduce evaporation
- Material, size, headspace, fill volume: Glass conical flasks (250mL) containing 100 mL test solution.
- Aeration: all flasks were incubated and shaken (approximately 100 rpm) in an orbital shaker
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: mean cell density at T=0h: 2.47 x10E4 cells/mL
Mean cell density values represent the mean number off cell per mL calculated from the mean of the cell counts from 3 fields of view for each or the replicate flasks.
- Control end cells density: Mean cell density at T=72h was 8.45 x 10E5 cells/mL
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: 3 replicates

TEST MEDIUM / WATER PARAMETERS
- Culture medium prepared according to OECD 201 requirement on test medium except for the concentration in
FeCl3.6H2O which was 0.08 mg/L instead of 0.064 as recommended (minor deviation)

OTHER TEST CONDITIONS
- Sterile test condition: no data
- Adjustment of pH: no data
- Photoperiod: continuous
- Light intensity and quality: approximately 7000 Lux. type of lamp used note recorded but if cool white fluorescent lamps used intensity is approx. 94.60 µ mol.m-2.s-1 which is conform to the OECD recommendation (between 60-120 µ mol.m-2.s-1).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: samples were taken at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm using a Jenway 6100 spectrophotometer. The cell densities of the control cultures at 0, 24, 48 and 72 hours, were determined by direct counting with the aid of a haemocytometer to confirm that the absorbance values were sufficiently well correlated with cell density values to be used to monitor the growth of the test cultures.
- Other: all test and control cultures were inspected microscopically at 72 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Tests concentrations: 0.625; 1.25; 2.5; 5.0; 10 mg/L
Reference substance (positive control):
not specified
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: EC50 (24-48h); P<0.001
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: P<0.001
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: P>= 0.05%
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities:
- Unusual cell shape: no
- Colour differences: at 72 hours there were no abnormalities detected in any of the control or test at 0.625; 1.25 and 5.0 mg/L. However at the test concentration of 100mg/L the cells were observed to be colourless.
Reported statistics and error estimates:
Statistical analysis of the area under the growth curve data was carried out using one way analysis of variance.
There were no statistically significant differences between the control 0.625; 1.25 and 2.5 mg/L test concentration (P>=0.05%), however all other test concentration were significantly different (P<0.001)

Table 2: Absorbance and pH values

Nominal
Concentration (mg/l)		 Absorbance values pH values
0h 24h 48h 72h 0h 72h
Control R1 0.031 0.065 0.318 0.821 8 10.3
  R2 0.03 0.067 0.347 0.881 8 10.2
  R3 0.03 0.062 0.344 0.857 8,1 10.2
  X 0.03 0.065 0.336 0.853    
0.625 mg/L R1 0.03 0.068 0.351 0.871 8 10.1
  R2 0.03 0.065 0.35 0.865 8,1 10.1
  R3 0.03 0.066 0.31 0.867 8 10.1
  X 0.03 0.066 0.337 0.868    
1.25 mg/L R1 0.031 0.066 0.324 0.833 8 10.2
  R2 0.03 0.061 0.339 0.831 8 10.3
  R3 0.03 0.065 0.37 0.862 8 10.1
  X 0.03 0.064 0.344 0.842    
2.5 mg/L R1 0.03 0.067 0.315 0.85 8 10.0
  R2 0.03 0.065 0.352 0.818 8 10.0
  R3 0.03 0.066 0.332 0.814 8 10.1
  X 0.03 0.066 0.333 0.827    
5.0 mg/L R1 0.03 0.042 0.056 0.102 8 10.0
  R2 0.03 0.04 0.061 0.112 8 9.4
  R3 0.03 0.045 0.07 0.107 8 9.7
  X 0.03 0.042 0.062 0.107    
10 mg/L R1 0.03 0.035 0.04 0.051 8 8.6
  R2 0.03 0.033 0.041 0.05 8 8.8
  R3 0.03 0.034 0.039 0.05 8 8.6
  X 0.03 0.034 0.04 0.05    

Table 3: Inhibition of growth

Nominal Concentration
 (mg/L)		 Area under
curve at 72 hour		 % inhibition Growth rate
(24-48h)		 % inhibition
Control 18.06   0.068  
0.625 18.288 -1 0.068 0
1.25 18.096 0 0.07 -3
2.5 17.7 2 0.067 1
5 1.98 89 0.016 76
10 0.576 97 0.007 90

From the data given in tables 2 and 3 it is clear that both the growth (r) and the biomass (b) of Scenedesmus subspicatus were affected by the presence of the test material over the 72 hour exposure period.

Toxicity results were determined from figure 3 (cf attached documents):

- EbE50 (72h)= 4.0 mg/L

- ErC50 (24 -48 h)= 4.6mg/L

Validity criteria fulfilled:
not specified
Remarks:
for details see freetext overall remarks
Conclusions:
In the test condition,phosphinic acid shows toxicity to Scenedesmus subspicatus but the tests shows important deviations to the guideline. As a consequence the test is considered as invalid for assessment.
Executive summary:

In a 72 hours acute toxicity study, the cultures of Scenedesmus subspicatus (CCAP 276/20) were exposed to phosphinic acid at nominal concentrations of 0 ; 0.625 ; 1.25 ; 2.5 ; 5.0 and 10 mg/L under static conditions in accordance with the OECD guideline 201. 

the toxicity results observed were:

based on biomass (area under the curve):

EbC50 -72h = 4.0 mg/L

NOEC -72h = 2.5 mg/L

based on growth rates from 24 -48h

ErC50 -48h= 4.6mg/L

The % growth inhibition in the treated algal culture as compared to the control ranged from -1 to 97 based on biomass results (area under the curve) and from -3 to 90 based on growth rates.

No abnormalities were noted at concentrations of 0 ; 0.625 ; 1.25 ; 2.5 ; 5.0 mg/L however at 10mg/L the cells were colourless.

However a detailed assessment of the study shows that some validity criteria are not fulfilled:

-The increase in cell density measured in the control between the end and the beginning was equivalent to a factor of 34(guideline requests >16).

- pH in control cultures should not increase by more than 1.5 units during the test. In this study pH in the control vary from 8 at T:0h to 10.3 (2.3 units of increase).

-The initial biomass concentration is 10 time greater than recommended

Moreover, some validity criteria cannot be assessed because of lack of information:

- The mean coefficient of variation for section-by-section specific growth rate in the control cultures is below the limit of 35%

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures is below the limit of 7%

But the report does not mention the raw data on cell density for each sample therefore they cannot be estimated from this study.

- The final concentrations of the substance should be maintained within the designated limit of 80% of the initial concentration in non-inoculated flasks. As no measured concentrations are recorded in this study this parameter cannot be verified.

Applicant's conclusion:

Considering pH deviation, no data on pH adjustment is indicated in the report and a deviation of the pH was observed in both control and test vessels. However, no statistically significant differences between the growth curves of the control and the test concentrations (P>=0.05%) was observed. This can be explained because both controls and test vessels showed a similar exponential growth. However initial biomass was not fulfilling OECD recommendations and may have affected the test results similarly in the control and the tests vessels.

In the test condition, phosphinic acid shows toxicity to Scenedesmus subspicatus but the tests shows important deviations to the guideline. As a consequence the test is considered as invalid for assessment.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2009-09-11 till 2009-09-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study following OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test) and EU method C.3 (Algal Inhibition Test). Study was GLP certified.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
concentrations of the test item were determined in the duplicate test medium samples from the highest nominal test concentrations (100 mg/L). Samples from the nominal test concentrations of 4.6 to 46 mg/L were not analyzed, as these concentrations were below the NOEC determined in this test).
From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times

- Sampling method: additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 15 mL) was too small to perform the analyses.

- Sample storage conditions before analysis: samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions. Furthermore, samples were protected from light until analysis.

No more data available
Vehicle:
no
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines

ACCLIMATION
- Acclimation period: inoculum culture was set up three days before the start of the exposure
- Culturing media and conditions (same as test or not): yes, algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions

No more data available
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
0.24 mmol/L (= 24 mg/L as CaCO3) (calculated value)
Test temperature:
22-23 °C
pH:
Start of the test: 8.0-8.1
End of the test: 8.8-9.2
Dissolved oxygen:
No data
Nominal and measured concentrations:
4.6, 10, 22, 46 and 100 mg/L (nominal concentrations)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open, but covered with a glass dish
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks
- Initial cells density: 10000 cells/mL (nominal); selected according to the recommendations of the OECD test guideline
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six


GROWTH MEDIUM
- Standard medium used: yes, according to the test guideline


TEST MEDIUM / WATER PARAMETERS
- Preparation of test medium: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 51.88 mg of the test item completely in 519 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Light intensity and quality: mean measured light intensity for the test solutions was approximately 7200 Lux (range: 6600 to 7660 Lux, n=9). Light intensity over incubation area (n=9) was within ±15% from the average light intensity (as recommended by the guideline)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A small volume of the algal suspension was withdrawn daily (24h, 48h and 72h) from each test flask for the measurement of the biomass and was not replaced. The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate.
- Other:
Sample was taken from the control and from the test concentration of nominal 100 mg/L to determine a potential influence of the test item on the algal cells
Shape and size of the algal cells were visually inspected
The mean values for growth rate and yield were calculated for each treatment


TEST CONCENTRATIONS
- Spacing factor for test concentrations: appr. 2.17
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: % Inhibition after 72 hours was 2.0, -3.5 and 8.3 for 1.0, 10 and 100 mg/L respectively
- Test medium appearance: for all concentrations a clear solution


No more data available
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes (see table 1 in remarks on results section)
- Observation of abnormalities (for algal test): no differences in shape and size of the algal cells was observed
- Any stimulation of growth found in any treatment: no (see table 2 in remarks on results section)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period

No more data available
Results with reference substance (positive control):
- Results with reference substance valid: yes, the result of the latest positive control test was within the internal historical range (72h EC50 for growth rate from 2000 to 2009 = 0.71-1.74 mg/L)
- EC50 (72h) for the growth rate: 1.1 mg/L (study C36893; performed in February 2009)
- Other:
potassium dichromate (positive control) is tested twice a year to demonstrate satisfactory test condition

No more data available
Reported statistics and error estimates:
The determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Dunnett’s tests

- The measured concentrations of the test item in the test media of the test concentration of 100 mg/L were 106 and 104% of the nominal values at the start and the end of the test, respectively. Hence the biological results are considerd related to the nominal concentrations of the test item.

- The test item had no significant inhibitory effect on the growth of the algae (average growth rate and yield; table 2 and 3) during the test period of 72 hours up to and including the highest nominal test concentration of 100 mg/L (results of Dunnett’s tests, one-sided, α = 0.05, Table 2 and Table 3). Hence, the 72-hour EC10, EC20 and EC50 values of the test item could not be determined

- A dose-response-relationship could also not be observed.

- Table 1: Biomass of algae in all treated groups for all examined time points

Nominal test item concentration

(mg/L)

Rep. n°.

Biomass of algae*

24 hours

48 hours

72 hours

Control

1

14.8

91.0

385.5

2

15.7

100.7

341.5

3

14.1

101.1

447.9

4

14.4

94.7

435.2

5

14.7

104.0

449.0

6

14.6

97.0

452.3

Mean

14.7

98.1

418.6

SD

0.5

4.8

45.2

4.6

1

12.4

78.1

285.7

2

14.1

92.4

430.0

3

13.9

98.6

319.3

Mean

13.5

89.7

345.0

SD

0.9

10.5

75.5

10

1

13.1

88.2

297.6

2

12.6

88.0

424.8

3

12.4

91.9

339.2

Mean

12.7

89.4

353.9

SD

0.3

2.2

64.9

22

1

13.2

81.5

376.7

2

12.0

86.0

433.9

3

12.4

94.3

351.2

Mean

12.5

87.3

387.3

SD

0.6

6.5

42.4

46

1

12.0

83.7

299.6

2

11.6

92.6

432.1

3

12.4

99.7

404.0

Mean

12.0

92.0

378.6

SD

0.4

8.0

69.9

100

1

11.0

82.8

373.5

2

13.2

95.5

346.8

3

12.8

96.7

421.2

Mean

12.4

91.7

380.5

SD

1.2

7.7

37.7

* biomass determined by fluorescence measurement (at least duplicate measurements per replicate) and given as relative fluorescence units (x 103). (the initial cell density of 10000 algal cells/mL corresponds to 3.3 x 103 relative fluorescence units)

- Table 2: Average growth rates (μ) in all treated groups for all examined time points

Nominal test item concentration

 

(mg/L)

Average growth rate μ (day-1) and inhibition of μ (Ir)

0-24 h

24-48 h

48-72 h

μ

Ir(%)

μ

Ir(%)

μ

Ir(%)

Control

1.50

0.0

1.70

0.0

1.62

0.0

4.6

1.41

5.8

1.65

2.7

1.55

4.2

10

1.36

9.7

1.65

2.7

1.56

3.6

22

1.34

10.7

1.64

3.5

1.59

1.6

46

1.30

13.6

1.67

1.9

1.58

2.2

100

1.32

11.8

1.66

2.0

1.58

1.9

- Table 3: Average yield (Y) in all treated groups for all examined time points

Nominal test item concentration

 

(mg/L)

Yield Y (x 103) and inhibition of Y (Iy)

0-24 h

24-48 h

48-72 h

Y

Iy

Y

Iy

Y

Iy

Control

11.4

0.0

94.8

0.0

415.3

0.0

4.6

10.2

10.6

86.5

8.8

341.7

17.7

10

9.4

17.5

86.1

9.2

350.6

15.6

22

9.2

19.1

84.0

11.4

384.0

7.5

46

8.7

23.7

88.7

6.4

375.3

9.6

100

9.1

20.5

88.4

6.8

377.2

9.2
Validity criteria fulfilled:
yes
Remarks:
see overall remarks section
Conclusions:
The result of the toxicity to aquatic algae of the sodium phosphinate was used to determine the toxicity of phosphinic acid as it is commercially prepared as the result of pH adjustment of the sodium hypophosphite (salt).
The influence of sodium phosphinate on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72h static test according to the OECD Guideline 201 and the EU Method C.3. The test substance had no significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 h up to and including the highest nominal test concentration of 100 mg/L. As a result the EC10, EC20, EC50 and LOEC values for growth rate and yield are higher than 100 mg/L. Furthermore no differences in shape and size of the algal cells was observed. Hence, the NOEC of the test substance sodium phosphinate and the phosphinic acid are at least equal to or higher than 100 mg/L for growth rate and yield.
Executive summary:

The result of the toxicity to aquatic algae of the sodium phosphinate was used to determine the toxicity of phosphinic acid as it is commercially prepared as the result of pH adjustment of the sodium phosphinate (salt).

The influence of sodium phosphinate on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72h static test according to the OECD Guideline 201 and the EU Method C.3. The nominal concentrations of the test substance of 4.6, 10, 22, 46 and 100 mg/L were tested (three replicas/concentration) in parallel with a control (six replicas). The measured concentrations of the test substance in the test media of 100 mg/L were 106 and 104% of the nominal values at the start and the end of the test, respectively. Thus the biological results were related to the nominal concentrations of the test item. Furthermore the test substance was stable in the test media over the test period of 72 hours.

The test item had no significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the highest nominal test concentration of 100 mg/L. As a result the 72h EC10, EC20, EC50 and LOEC values for growth rate and yield are higher than 100 mg/L. Furthermore no differences in shape and size of the algal cells was observed. Hence, the NOEC (72h) of the test substance sodium phosphinate and the phosphinic acid are at least equal to or higher than 100 mg/L for growth rate and yield.

These results indicate that sodium phosphinate and its parent compound the phosphinic acid should be considered non-hazardous to the aquatic environment according to the CLP regulation (EC) N° 1272/2008 as the EC50(72h) for growth rate and yield is > 100 mg/L.

This study is GLP certified guideline study following OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test) and EU method C.3 (Algal Inhibition Test) and it meets all of the validity criteria (see overall remarks section). Thus we consider it a Klimisch 1 study

Description of key information

One key study is available for phosphinic acid on the growth of the green alga Desmodesmus subspicatus according to the OECD Guidelines No 201 and EU C.3 of Commission Regulation (EC) No 761/2009 and GLP compliant. As a result the 72h EC50 and EC10 on growth rate are > 160 mg/L and 98 mg/L respectively. These results are supported by a test performed on the sodium phosphinate according to OECD 201 with GLP compliance and allow to disregarded another study performed on the same algae species according to the OECD 201 with major deviations and without GLP statement.
Under the CLP regulation (EC) N° 1272/2008, these results lead to the conclusion that phosphinic acid is non hazardous to the aquatic environment and should not be classified for the environment.

Key value for chemical safety assessment

EC50 for freshwater algae:
160 mg/L
EC10 or NOEC for freshwater algae:
40 mg/L

Additional information

One key study and one disregarded study are avalaible on phosphinic acid.

The disregarded study on phosphinic acid was performed on Scenedesmus subspicatus with nominal concentrations of 0; 0.625; 1.25; 2.5; 5.0 and 10 mg/L under static conditions in accordance with the OECD guideline 201. The toxicity results observed were 48h ErC50 = 4.6mg/L based on growth rate. The study was not performed under GLP and in the absence of information on raw biomass data, concentrations over the test and pH adjustment, we cannot excluded that important deviations (pH variation, initial biomass and cell density) may have affected the results of the test. Therefore a a new study was performed on the same algae species and the results lead to disregarded this study.

The key study was performed according to OECD 201 and under GLP on Desmodesmus subspicatus with phosphinic acid. Tests conditions were exactly the same as in the previous study except that pH was adjusted and particular attention was put on all possible deviations. All test concentrations were corrected for the test item water content of 50%. Due to the acidic nature of the test item, the pH of the test preparations was adjusted to pH 7.5 prior to inoculation with algal test. As a result the 72h EC50, EC10 and NOEC values for growth rate are > 160, 98 and 40 mg/L, respectively. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 87% to 92% of nominal and so the results are based on nominal test concentrations only.

A supportive study was performed with sodium phosphinate on Pseudokirchneriella subcapitata in a 72h static test according to the OECD Guideline 201. Phosphinic acid is commercially prepared as the result of pH adjustment of the sodium phosphinate salt. The main assumption is that in aqueous solution the acid and the salt will be fully dissociated and the sodium cation is not significant in respect to effects on algae which are expected to be related to the phosphinate anion. Hence the phosphinic acid properties can be directly read across from its salt, the sodium phosphinate.

Nominal concentrations of 0, 4.6, 10, 22, 46 and 100 mg/L were tested and the measured concentrations of the test substance in the test media were 106 and 104% of the nominal values at the start and the end of the test, respectively. Thus the biological results were related to the nominal concentrations of the test item. The substance tested had no significant inhibitory effect on the growth of the algae with the 72h EC10, EC50 and LOEC values for growth rate higher than 100 mg/L confirming the disregarding of the Safepharm study (1995).

These results on algae indicate that phosphinic acid should be considered non-hazardous to the aquatic environment according to the CLP regulation (EC) N° 1272/2008.