Phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters, salts with 2,2'-iminodiethanol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Remarks:

See the Justification of Read Across

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Read Across justification document

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

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, mixed decyl and octyl esters, compds. with diethanolamine
CAS No.: 68425-57-0
Physical state: white solid at 20 °C (turbid, non-pourable paste)
Batch No.: PU61810016
Re-certification date of batch: 16 June 2018
Purity: 100 % (UVCB)
Moisture, % (KF) 1,2
pH, 10% in dW 7,2
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.

Vehicle:

no

Details on test solutions:

The medium used for the test was AAP-Medium (according to Annex 3 of OECD 201. 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: 22.8 – 23.2 °C

pH:

pH of control: 7.47 – 7.64

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 measured initial concentration of analyte ranged from 97 % to 102 % of nominal. In the aged samples the measured content was between

Details on test conditions:

Test procedure: Dose response test (static)
Duration: 72 hours
Temperature: 22.8 – 23.2 °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: 7.47 – 7.64
Illumination: Continuously from the side, 88.8 µEm-2s-1 (mean)
Initial cell density: 0.5 x 104 cells/mL (nominal)
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:

EC50

Effect conc.:

32.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

30.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

29.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Results with reference substance (positive control):

Potassium dichromate is tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions. The EC50 values calculated in this reference test were considered to be within an acceptable range therefore it can be considered that the test organism is sensitive.

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 (Jonckheere Terpstra test, left sided, for growth rate and yield). 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 above 100 % were set to 100 and values below 0 % were set to zero. Only concentrations within a clear concentration response relation were used for calculations.

Results

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 42.60 for the control, which exceeds the threshold of 16. It corresponds to a growth rate of 1.24032 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 31 % 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 7 % for the control and did not exceed 7 % for the whole test period.

Biological Results

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 9.77 mg/L. At higher test item concentrations the inhibition of growth rate peaked in 122.2 % at a nominal concentration of 100 mg/L and the inhibition of yield peaked in 100.8 % 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. Fewer cells were observed at 9.77 mg/L. 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. The mean light intensity of all positions of the incubator was 88.8 µEm-2s-1 with mean values of each platform in a range from 82.6 to 93.6 µEm-2s-1 (-7.0 % to +5.4 % of mean) which was within ± 15 % of variation as specified by OECD 201.

Analytical Results: The measured initial concentration of analyte ranged from 97 % to 102 % of nominal. In the aged samples the measured content was between <LOQ and 100 % of nominal. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item.

Table: Percentage inhibition of growth rate

Conc. [mg/L]	% Inhibition of growth rate
	0 h - 24 h	0 h - 48 h	0 h - 72 h
Control	0.0	0.0	0.0
0.954	-29.5	-25.3	-20.9 (1)
3.05	-32.7	-29.6	-24.3 (1)
9.77	28.3	-0.6	-0.1 (2)
31.3	107.6	24.9	32.9 *
100	168.1	122.4	122.2 *(3)

1) Value was omitted from statistical evaluation

2) Value was set to zero for EC10, 20, 50-calculation

3) Value was set to 100 for EC10, 20, 50-calculation

* Statistically significant different to the control

Table: Percentage inhibition of yield

Conc. [mg/L]	% Inhibition of yield
	0 h - 24 h	0 h - 48 h	0 h - 72 h
Control	0.0	0.0	0.0
0.954	-51.4	-77.6	-117.8 (1)
3.05	-58.1	-93.9	-142.9 (2)
9.77	37.8	-0.3	2.3
31.3	104.1	40.9	72.8 *
100	128.4	104.2	100.8 *(3)

1) Value was omitted from statistical evaluation

2) Value was set to zero for EC10, 20, 50-calculation and omitted from NOEC-calculation

3) Value was set to 100 for EC10, 20, 50-calculation

* Statistically significant different to the control

Table: Determined concentration of the test item

test item - nominal	Sampling	test item found
[mg/L]		[mg/L]	% of nominal
0	0 h fresh 72 h aged	<LOQ <LOQ	n.d. n.d.
0.954	0 h fresh 72 h aged	0.958 <LOQ	100 n.d.
3.05	0 h fresh 72 h aged	3.03 2.90	99 95
9.77	0 h fresh 72 h aged	9.60 9.08	98 93
31.3	0 h fresh 72 h aged	32.0 31.4	102 100
100	0 h fresh 72 h aged	96.6 97.1	97 97

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 31.3 mg/L (nominal) and above. The overall LOEC was therefore determined to be 31.3 mg/L (nominal), the corresponding NOEC was set at 9.77 mg/L (nominal). The EC10-value for growth rate (ErC10) was determined to be 29.5 mg/L (nominal). The EC10-value for yield (EyC10) was 15.2 mg/L (nominal). The EC20-value for growth rate (ErC20) was determined to be 30.5 mg/L (nominal). The EC20-value for yield (EyC20) was 18.9 mg/L (nominal). The EC50-value for growth rate (ErC50) was determined to be 32.1 mg/L (nominal). The EC50-value for yield (EyC50) was 26.1 mg/L (nominal).

Executive summary:

The objective of this study was to determine the effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata, to determine the no observed effect concentration (NOEC), to determine the lowest observed effect concentration (LOEC) and to determine the effect concentration (EC10, 20, 50), where possible. Principles were the exposure of algae to test solutions and the determination of cell division after 24, 48 and 72 hours of exposure, in order to evaluate a potential inhibition of the cell multiplication. The study was performed according to OECD test guideline 201 (2011).

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 31.3 mg/L (nominal) and above. The overall LOEC was therefore determined to be 31.3 mg/L (nominal), the corresponding NOEC was set at 9.77 mg/L (nominal). The EC10-value for growth rate (ErC10) was determined to be 29.5 mg/L (nominal). The EC10-value for yield (EyC10) was 15.2 mg/L (nominal). The EC20-value for growth rate (ErC20) was determined to be 30.5 mg/L (nominal). The EC20-value for yield (EyC20) was 18.9 mg/L (nominal). The EC50-value for growth rate (ErC50) was determined to be 32.1 mg/L (nominal). The EC50-value for yield (EyC50) was 26.1 mg/L (nominal).

Description of key information

The toxicity to algae was evaluated with results of a similar substance tested according to the OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test) and EU Method C.3 (Algal Inhibition test). See the justification of Read Across.

Key value for chemical safety assessment

EC50 for freshwater algae:

32.1 mg/L