Phosphoric acid, mono- and di-C6-12-(even numbered)-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:

From January 02, 2018 to January 05, 2018

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The total exposure period was 72 h and samples for analytical confirmation of actual exposure concentrations were taken at the start and at the end of exposure.

Vehicle:

yes

Remarks:

water

Details on test solutions:

Test substance was a white solid (UVCB) and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test substance.
Preparation of test solutions started with loading rates individually prepared at loading rates of 1.0 to 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test substance in test medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test solutions. All test solutions were clear and colorless at the end of the preparation procedure except for the highest test concentration in the combined limit/range-finding test which was clear but slightly light yellow.

Test organisms (species):

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

Details on test organisms:

- Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Reason for selection: This system is a unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae Culture
Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium:
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium:
M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

21 to 23°C

pH:

8.2 to 8.5

Details on test conditions:

Loading concentrations: 10, 18, 32, 56 AND 100 mg/L
0 h measured concentrations: 3.5, 7, 12, 19 and 30 mg/L
72 h measured concentrations: 2.8, 4.6, 7.6, 12 and 18 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: growth inhibition and yeild

Details on results:

A study was conducted to determine the short term toxicity of the substance to aquatic algae and cyanobacteria according OECD Guideline 201, in compliance with GLP. Pseudokirchneriella subcapitata were exposed to a series ofWater Accommodated Fractions (WAFs) were individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L and used as test concentrationsof the substance for 72 h. Total organic carbon (TOC) concentrations were measured with a TOC analyser and the test substance was considered stable over the duration of the test.Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. The average growth rate as well as the percent of inhibition of growth rate were calculated. The NOEL of the test substance for growth rate and yield< 10 mg/Lbased on statistical significance however at 10 mg/L concentration there was no biological significance. Under the study conditions, the 72 h EL50 for growth rate and yield are54 mg/L and 19 mg/L,respectively (Augusiak, 2018).

Results with reference substance (positive control):

Reference substance inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.

The EC50 for growth rate inhibition (72h-ERC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.34 mg/L with a 95% confidence interval ranging from 0.34 to 0.35 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.

Combined limit/range finding test:

At the end of the test, the replicates of the highest test concentration had turned slightly hazy and 36% growth rate inhibition and 86% yield inhibition were observed at this concentration level. The expected EL₅₀for growth rate inhibition was above a WAF prepared at a loading rate of 100 mg/L. The expected EL₅₀for inhibition of yield was between WAFs prepared at loading rates of 10 and 100 mg/L.

Samples taken from WAFs prepared at loading rates of 10 and 100 mg/L were analysed.The initial TOC concentrations were 2.3 and 14 mg/L, respectively, corresponding to 29-49% of the nominal TOC concentrations as based on the loading rates. The lower concentration remained stable during the test period, i.e., was 103% of initial after 72 hours. The higher concentration decreased to 66% of the initial concentration during the test.

All test conditions were maintained within the limits prescribed by the study plan.Except for the temperature during the first two hours of the test, which deviated by more than 2°C and varied between 21 and 23°C. However, the validity criteria of the test were met, indicating that this temperature deviation had no effect on the results.

Mean Cell Densities (x10⁴Cells/mL) During the Combined Limit/Range-Finding Test

Time (h)	0312 PHOSPHORSÄUREPARTIALESTER K-SALZ C8-10 WAF loading rate (mg/L)
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	7.5	n.d.	n.d.	5.9
48	39.7	n.d.	n.d.	10.2
72	210.9	250.7	253.5	31.2

n.d.: not determined.

Percentage Inhibition of Growth Rate During the Combined Limit/Range-Finding Test

Test substance WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.783	0.0149	6
1.0	1.841	0.0132	3	-3.2
10	1.845	0.0117	3	-3.5
100	1.141	0.0690	6	36

Percentage Inhibition of Yield During the Combined Limit/Range-Finding Test

Test substance WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	209.9	9.58	6
1.0	249.7	9.83	3	-19
10	252.5	9.00	3	-20
100	30.2	6.51	6	86

Final test:

Measured TOC concentrations:            

Samples taken from all test groups were analysed. The TOC concentrations measured increased with increasing loading rate, indicating an appropriate preparation of the test solutions. The initial TOC concentrations corresponded to 63-82% of the nominal TOC concentrations as based on the applied loading rates. The measured TOC concentrations decreased to 60-82% of the initial concentrations at the end of the test.

Since TOC-analysis is a non-specific method, the effect parameters were determined based on loading rates.

Measured TOC Concentrations Versus Nominal TOC Concentrations

Test substance	Measured concentration (TOC, mg/L)		% relative to nominal
WAF loading rate (mg/L)	t=0h	t=72 h	at t=0h
10 (4.7)	3.5	2.8	73
18 (8.5)	7	4.6	82
32 (15)	12	7.6	80
56 (26)	19	12	71
100 (47)	30	18	63

( ): Nominal TOC concentration in mg/L (values based on applied loading rate).

 

Below table shows group mean growth rates and the percentages of growth rate inhibition (total test period), whereasTable6shows the values at different time intervals.

Growth rates of algae exposed to WAFs prepared at 10 mg/L and higher were increasingly reduced and the inhibition of growth rate was statistically significant at all test concentrations. The inhibition of growth rate observed at a loading rates of 10 mg/L was below 10% and thus considered to be biologically not relevant.

Inhibition of yield increased with increasing loading rate from 10 mg/L upwards resulting in 97-98% inhibition at the two highest loading rates. Statistically significant inhibition of yield was found at all test concentrations.

The test solutions of the three highest test concentrations had turned hazy after 48 hours of exposure and remained hazy until the end of the test.

A slightly lower inhibition of both growth rate and yield was observed at the highest test concentration compared to the WAF prepared at a loading rate of 56 mg/L. Cell growth varied more strongly among the replicates of the 56 mg/L test group than in the 100 mg/L test group. The reason for this could not be specified but it is possible that the hazy test solutions had a small impact on algal growth. This had, however, no negative impact on the conclusions or the integrity of the test. 

Microscopic observations at the end of the test revealed a normal and healthy appearance of algal cells exposed to a WAF prepared at a loading rate of 18 mg/L when compared to the control.

Growth Rate And Percentage Inhibition For The Total Test Period

Test substance WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.888	0.0058	6
10	1.851	0.0096	3	1.9*^#
18	1.680	0.0288	3	11*
32	1.306	0.2039	3	31*
56	0.631	0.1915	3	67*
100	0.758	0.0331	3	60*

* effect statistically significant;^#effect biologically not relevant (<10%).

Growth Rate And Percentage Inhibition At Different Time Intervals

Test substance WAF loading rate (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.287		1.826		1.549
10	3	2.333	-2.0	1.699	7.0	1.521	1.8
18	3	2.128	7.0	1.358	26	1.553	-0.2
32	3	1.670	27	0.839	54	1.409	9.1
56	3	1.160	49	1.410	23	-0.676	144
100	3	0.202	91	2.044	-12	0.029	98

Yield And Percentage Inhibition For The Total Test Period

Test substance WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	287.0	4.95	6
10	257.0	7.40	3	10*
18	153.7	13.09	3	46*
32	55.0	27.12	3	81*
56	6.4	3.84	3	98*
100	8.8	0.95	3	97*

* effect statistically significant;^#effect biologically not relevant (<10%).

Effect Parameters

	Parameter (mg/L)	NOEL*	NOEL**	EL10	EL20	EL50
Growth rate	Value	<10	10	13	22	54
	lower 95%-cl			10	18	49
	upper 95%-cl			16	25	60
Yield	Value	<10	<10	9.5	12	19
	lower 95%-cl			8.8	11	19
	upper 95%-cl			10	13	20

cl: confidence limit; * based on statistical significance; ** based on biological relevance.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h EL50 for growth rate and yield are 54 mg/L and 19 mg/L,respectively.

Executive summary:

A study was conducted to determine the short term toxicity of the substance to aquatic algae and cyanobacteria according OECD Guideline 201, in compliance with GLP. Pseudokirchneriella subcapitata were exposed to a series of Water Accommodated Fractions (WAFs). These WAFs were individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L and used as test concentrations for 72 h. Total organic carbon (TOC) concentrations were measured with a TOC analyser and the test substance was considered stable over the duration of the test. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. The average growth rate as well as the percent of inhibition of growth rate were calculated. The NOEL of the test substance for growth rate and yield <10 mg/L based on statistical significance; however at 10 mg/L concentration there was no biological significance. Under the study conditions, the 72 h EL50 for growth rate and yield are 54 mg/L and 19 mg/L, respectively (Augusiak, 2018).

Key value for chemical safety assessment

EC50 for freshwater algae:

54 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

A study was conducted to determine the short term toxicity of the substance to aquatic algae and cyanobacteria according OECD Guideline 201, in compliance with GLP. Pseudokirchneriella subcapitata were exposed to a series of Water Accommodated Fractions (WAFs). These WAFs were individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L and used as test concentrations for 72 h. Total organic carbon (TOC) concentrations were measured with a TOC analyser and the test substance was considered stable over the duration of the test. Since TOC-analysis is a non-specific method, the effect parameters were reported in terms of loading rates initially prepared. The average growth rate as well as the percent of inhibition of growth rate were calculated. The NOEL of the test substance for growth rate and yield <10 mg/L based on statistical significance; however at 10 mg/L concentration there was no biological significance. Under the study conditions, the 72 h EL50 for growth rate and yield are 54 mg/L and 19 mg/L,respectively (Augusiak, 2018).