Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol]

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 November 10, 2017 to December 17, 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:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The total exposure period was 72 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure.

Vehicle:

yes

Remarks:

Water (WAF)

Details on test solutions:

Test substnace was a white turbid liquid (UVCB) and not completely soluble in test medium at the loading rates initially prepared. A correction was made for the water content of the test substance. A correction factor of 4.99 was used, i.e. the nominal amount to be tested was multiplied with the correction factor to determine the amount of test substance to be weighed for the test solution preparation. Concentrations are expressed as “mg/L” when referring to the tested fraction of the test substance.

Preparation of test solutions started with loading rates individually prepared at loading rates of 1.0 to 100 mg UVCB/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test substance in test medium. The obtained mixtures were allowed to stabilize overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were siphoned off over glass wool and used as test solutions. Test solutions prepared at 56 mg/L and higher were slightly hazy, whereas lower test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

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

Hardness:

24 mg CaCO3/L

Test temperature:

22 to 23°C

pH:

7.7 to 9

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100 mg/L
at 0h measured concentrations: 4.4, 7.2, 12,21 and 20 mg/L
at 72 h measured concentrations: 3.5, 6.5, 11, 17 and 29 mg/L

Details on test conditions:

Combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a WAF prepared at a loading rate of 100 mg/L.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• Three replicates per concentration were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L.
• pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed to verify a normal and healthy appearance.
• At each loading rate, two replicates without algae were pooled before sampling at the end of the test.
• Samples of 40-50 mL were taken from all test concentrations for possible TOC analysis.
• At the beginning of the test, cells were counted using a microscope and a counting chamber. Cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

Test concentrations:
Test substance: WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg UVCB/L
Controls: Test medium without test substance or other additives.
Replicates:
3 replicates of each test concentration,
6 replicates of the control,
2 extra replicates of each test group without algae for sampling purposes at the end of the test,
1 replicate of each test concentration without algae.

Test procedure and conditions
Test duration: 72 hours
Test type: Static
Test vessels 100 mL, all-glass, containing 50 mL of test solution
Medium M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements and recordings
- pH At the beginning and at the end of the test.
- Temperature of medium Continuously in a temperature control vessel.

- Appearance of the cells At the end of the final test, microscopic observations were performed on the control and the WAF prepared at 56 mg/L to observe for any abnormal appearance of the algae.

Recording of Cell Densities
At the start of the final test, undissolved particles were observed in the highest test concentration that disturbed spectrophotometric measurement. Therefore algal density was determined by use of a microscope and a counting chamber throughout the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

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:

growth rate

Remarks on result:

other: based on statistical significance

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: based on biological significance

Results with reference substance (positive control):

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.

Results:

Combined limit/tange finding test:

At the end of the test, 15% growth rate inhibition and 57% inhibition of yield were observed. The expected EL₅₀for growth rate inhibition was above a WAF prepared at a loading rate of 100 mg/L. The expected EL₅₀for 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 3.1 and 30 mg/L, respectively, corresponding to 49-51% of the nominal TOC concentrations as based on the loading rates. The lower concentration remained stable during the test period, i.e., was 97% of initial after 72 h. The higher concentration decreased to 73% of the initial concentration during the test. All test conditions were maintained within the limits prescribed by the study plan.

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

Time (h)	test substance (mg/L)
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	9.2	n.d.	n.d.	7.7
48	56.3	n.d.	n.d.	35.5
72	274.4	285.5	275.0	118.2

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	1.871	0.0164	6
1.0	1.885	0.0085	3	-0.72
10	1.872	0.0260	3	-0.02
100	1.588	0.0495	6	15

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

Test substance WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	273.4	13.36	6
1.0	284.5	7.36	3	-4.1
10	274.0	21.28	3	-0.20
100	117.2	17.21	6	57

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 20-73% of the nominal TOC concentrations as based on the applied loading rates. The WAF prepared at a loading rate of 100 mg/L showed a similar TOC concentration as the WAF prepared at a loading rate of 56 mg/L, indicating that the maximum solubility of test substance in test medium was most likely reached at these loading rates. The TOC concentrations measured at the end of the test had remained mostly stable, i.e. were at 79-92% of the initial concentrations in WAFs prepared at 10-56 mg/L. The WAF prepared at 100 mg/L was at 144% of the initially measured TOC concentration at the end of the test. A reason for this increase in measured TOC could not be identified.

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^1 WAF loading rate (mg/L)	Measured concentration (TOC, mg/L)		% relative to nominal at t=0h
	t=0h	t=72 h
10 (6.1)	4.4	3.5	73
18 (11)	7.2	6.5	40
32 (20)	12	11	37
56 (34)	21	17	37
100 (61)	20	29	20

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

Inhibition of growth rate and inhibition of yield:

A dose-related increase of inhibition of growth rate and yield was observed at all loading rates. Statistically significant inhibition of both growth rate and yield was observed at loading rates of 18 mg/L and higher. The inhibition of growth rate observed at loading rates of 18 and 32 mg/L were below 10% and thus considered to be biologically not relevant.Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a WAF prepared at 56 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	1.684	0.0470	6
10	1.660	0.0199	3	1.4
18	1.620	0.0529	3	3.8*^#
32	1.519	0.0210	3	9.8*^#
56	1.333	0.0067	3	21*
100	1.248	0.0763	3	26*

* Effect was statistically significant.;^#Effect was 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	1.834		1.576		1.642
10	3	1.404	23	1.854	-18	1.722	-4.9
18	3	1.251	32	1.870	-19	1.738	-5.8
32	3	1.228	33	1.599	-1.5	1.731	-5.4
56	3	1.347	27	1.254	21	1.397	15
100	3	0.881	52	1.378	13	1.484	9.7

Yield And Percentage Inhibition For The Total Test Period

Test substance WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	156.7	21.49	6
10	144.7	8.81	3	7.7
18	129.0	21.27	3	18*
32	94.5	6.06	3	40*
56	53.5	1.09	3	66*
100	42.0	10.40	3	73*

* Effect was statistically significant.

Effect parameters:

	Parameter (mg/L)	NOEL*	NOEL**	EL10	EL20	EL50
Growth rate	Value	10	32	31	66	>100
	lower 95%-cl			26	59
	upper 95%-cl			36	73
Yield	Value	10	10	11	18	43
	lower 95%-cl			8.7	15	39
	upper 95%-cl			13	20	47

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 was determined to be 100 mg/L respectively.

Executive summary:

A study was conducted to determine the short term toxicity of the substance, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], to aquatic algae and cyanobacteria according to OECD Guideline 201, in compliance with GLP. Pseudokirchneriella subcapitata were exposed to a series of Water Accommodated Fractions (WAFs), 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 was detrmined to be 10 mg/L based on statistical significance and at 32 mg/L based on biological significance. Under the study conditions, the 72 h EL50 for growth rate was determined to be 100 mg/L (Augusiak, 2018).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 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, Phosphoric acid, mono- and di-decyl ester, compd. with 2,2',2''-nitrilotris[ethanol], to aquatic algae and cyanobacteria according to OECD Guideline 201, in compliance with GLP. Pseudokirchneriella subcapitata were exposed to a series of Water Accommodated Fractions (WAFs), 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 was detrmined to be 10 mg/L based on statistical significance and at 32 mg/L based on biological significance. Under the study conditions, the 72 h EL50 for growth rate was determined to be 100 mg/L (Augusiak, 2018).