Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Analytical monitoring:

yes

Details on sampling:

Analytical data are required by the guidelines for verification of test item concentrations as well as the stability of the test item over the entire test period. Analytical samples were taken at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions from all test concentrations and control. For each sampling also a retain sample was taken. 500 µL samples were taken and stabilized with 500 µL methanol.
All samples were stored deep frozen until they were transferred to the analytical laboratory.

Vehicle:

no

Details on test solutions:

The medium used for the test was AAP-Medium (according to Annex 3 of OECD 201, composition see Appendix B). The pH was adjusted to 7.5 +- 0.1 with NaOH or HCl, if necessary.

Test organisms (species):

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

Details on test organisms:

The test organism Pseudokirchneriella subcapitata Hindák (Sphaeropleales: Selenastraceae), strain: SAG 61.81, was purchased from a commercial supplier, e.g. MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany. The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from our commercial supplier.
Culture conditions are as follows:
- Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 – 120 µEm-2s-1)
- Temperature: 21 - 24 °C
- Culture flasks: 100 mL Erlenmeyer flasks
- CO2 supply by shaking on a rotating shaker, approximately 105 rpm

Cells from this semi-continuous liquid stock culture were used for the test. 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

The medium used for the test was AAP-Medium (according to Annex 3 of OECD 201, composition see Appendix B).

Test temperature:

Temperature: 22.5 – 23.0 °C

pH:

pH of control: 7.68 – 7.75

Salinity:

The medium used for the test was AAP-Medium (according to Annex 3 of OECD 201, composition see Appendix B).

Nominal and measured concentrations:

The analysed concentrations of fresh and aged test item solutions are presented below.

Test item nominal [mg/L] test item nominal [mg/L] Sampling test item found
[mg/L] % of nominal

Control 0 0 h fresh 0 n.d.
72 h aged 0 n.d.

0.954 0.954 0 h fresh 1.01 106
72 h aged
3.05 3.05 0 h fresh 3.02 99
72 h aged 1.08 35

9.77 9.77 0 h fresh 7.92 81
72 h aged 7.54 77

31.3 31.3 0 h fresh 32.0 102
72 h aged 34.0 109

100 100 0 h fresh 100 100
72 h aged 108 108

Details on test conditions:

Test procedure: Dose response test (static)
Duration: 72 hours
Temperature: 22.5 – 23.0 °C
CO2 supplied: By continuous agitation: Test vessels were placed in an incubator on a pivoted bogie which turns around and induces shaking by regular sudden stops
pH of control: 7.68 – 7.75
Illumination: Continuously from the side, 79.7 µEm-2s-1
Initial cell density: 0.5 x 10^4 cells/mL

The incubation took place in a temperature controlled light incubator. The incubator is equipped with a pivoted bogie consisting of several platforms. The bogie turns around passing laterally placed fluorescent tubes, which enables uniform lighting of the test vessels.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

7.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.707 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

9.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Results with reference substance (positive control):

Toxic Reference

In order to check the validity of the results, the toxicity of the reference item potassium dichromate was tested at 0.0512, 0.128, 0.320, 0.800 and 2.00 mg/l. The results after 72h exposure indicated an NOEC (nominal) of the reference item potassium dichromate of 0.320 mg/l, a LOEC (nominal) of 0.800 mg/l, an ErC10 (nominal) of 0.695 mg/l and an EyC10 of 0.354 mg/l. Since the results are in accordance with the requirements of the OECD guideline 201, the test organisms were suitable for the determination of the toxicological effects of the test item.

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided). The EC10, 20, 50-values for growth rate and yield were determined by probit analysis following the Gompertz distribution, respectively. Due to statistical reasons inhibition-values below 0 % were set to zero.

Validity Criteria of the Study

Biomass:

Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 32.48 for the control, which exceeds the threshold of 16. It corresponds to a growth rate of 1.15585 d-1.

Coefficient of Variation (section by section):

The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 18 % for the control and did not exceed 35 %.

Coefficient of Variation (average growth):

The coefficient of variation of average growth in replicate control cultures was 4.8 % for the control and did not exceed 7 % for the whole test period.

Main Test

After 72 h at termination of the test no statistically significant inhibition of growth rate or yield was observed for concentrations up to and including 3.05 mg/L. At higher test item concentrations the inhibition of growth rate peaked in 57.3 % at a nominal concentration of 100 mg/L and the inhibition of yield peaked in 88.9 % at a nominal concentration of 100 mg/L.

The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item concentration of 3.05 mg/L. A reduction of cells was observed at 9.77 mg/L test item concentration. No cells were observed at 31.3 and 100 mg/L nominal test item concentration.

The growth conditions (pH and temperature) during the test were within the range specified by OECD 201 guideline.

The mean light intensity of all positions of the incubator was 79.7 µEm-2s-1 with mean values of each platform in a range from 73.2 to 83.8 µEm-2s-1 (-8.2 % to +5.1 % of mean). The light intensity of all positions is measured once a year and was confirmed for one representative position at test start.

Analytical Results

The measured initial concentration of the test item ranged from 81 % to 106 % of nominal. In the aged samples the measured content was between < LOQ % and 109 % of nominal. Therefore toxicological endpoints were evaluated using the nominal concentrations (based on the geometric mean of each measured concentration) of the test item.

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 9.77 mg/L (nominal) and above. The overall LOEC was therefore determined to be 9.77 mg/L (nominal), the corresponding NOEC was set at 3.05 mg/L (nominal). The EC10-value for growth rate (ErC10) was determined to be 2.13 mg/L (nominal). The EC10-value for yield (EyC10) was 0.707 mg/L (nominal). The EC20-value for growth rate (ErC20) was determined to be 7.48 mg/L (nominal). The EC20-value for yield (EyC20) was 2.10 mg/L (nominal). The EC50-value for growth rate (ErC50) was determined to be 50.0 mg/L (nominal). The EC50-value for yield (EyC50) was 10.9 mg/L (nominal).

Executive summary:

In the Klimisch 1 GLP study the toxicity of the test item to algae was determined in a 72‑hour static test according to Part C.3 of the Commission Regulation (EC) No 440/2008 and the OECD Guideline for Testing of Chemicals No. 201.

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 9.77 mg/L (nominal) and above. The overall LOEC was therefore determined to be 9.77 mg/L (nominal), the corresponding NOEC was set at 3.05 mg/L (nominal).

The EC10-value for growth rate (ErC10) was determined to be 2.13 mg/L (nominal). The EC10-value for yield (EyC10) was 0.707 mg/L (nominal). The EC20-value for growth rate (ErC20) was determined to be 7.48 mg/L (nominal). The EC20-value for yield (EyC20) was 2.10 mg/L (nominal). The EC50-value for growth rate (ErC50) was determined to be 50.0 mg/L (nominal). The EC50-value for yield (EyC50) was 10.9 mg/L (nominal).

Description of key information

The EC10-value for growth rate (ErC10) of the test item was determined to be 2.13 mg/L (nominal). The EC10-value for yield (EyC10) was 0.707 mg/L (nominal). The EC20-value for growth rate (ErC20) was determined to be 7.48 mg/L (nominal). The EC20-value for yield (EyC20) was 2.10 mg/L (nominal). The EC50-value for growth rate (ErC50) was determined to be 50.0 mg/L (nominal). The EC50-value for yield (EyC50) was 10.9 mg/L (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:

50 mg/L

EC10 or NOEC for freshwater algae:

2.13 mg/L