Bis[2-[(2-aminoethyl)amino]ethanolato][2-[(2-aminoethyl)amino]ethanolato-O](propan-2-olato)titanate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 - 28 Apr 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2011)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Bis[2-[(2-aminoethyl)amino]ethanolato][2-[(2-aminoethyl)amino]ethanolato-O](propan-2-olato)titanate

EC Number:

265-723-8

EC Name:

Bis[2-[(2-aminoethyl)amino]ethanolato][2-[(2-aminoethyl)amino]ethanolato-O](propan-2-olato)titanate

Cas Number:

65380-84-9

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

2-methyl-4,4-bis(propan-2-yloxy)-3,5-dioxa-8-aza-4-titanadecan-10-amine; 7,7-bis(propan-2-yloxy)-6,8-dioxa-3,11-diaza-7-titanatridecane-1,13-diamine; 7,7-bis({2-[(2-aminoethyl)amino]ethoxy})-6,8-dioxa-3,11-diaza-7-titanatridecane-1,13-diamine; 7-{2-[(2-aminoethyl)amino]ethoxy}-7-(propan-2-yloxy)-6,8-dioxa-3,11-diaza-7-titanatridecane-1,13-diamine; tetrakis(propan-2-yloxy)titanium

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Test solutions were analyzed indirectly for concentrations of the analytical marker titanium via ICP-MS

Details on sampling:

- Concentrations: Each test substance and control at test initiation (0 h) and termination (72 h),
- Sampling method: 10 mL samples were collected using a plastic serological pipette and placed in separate 15-mL Falcon™ tubes. At 0 h samples were collected from parent solutions. At 72 h amples were collected after combining replicate solutions by treatment.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg KR®44/L test solution was prepared as a water accommodated fraction. 0.1000 g of KR®44 was weighed into a disposable polypropylene funnel and dropped into a glass carboy containing 1.0 L of test medium. The solution was stirred using a magnetic stir bar so that the vortex was no greater than ~25% of the solution depth. The solution was covered and stirred at room temperature for approximately 22 hours. The carboy opening was covered with foil. The solution was allowed to settle for approximately 2 hours, then approximately 200 mL was decanted from the carboy. Appropriate aliquots of the WAF solution were diluted in 0.5 L of test medium to produce the different test treatment solutions. The 100 mg KR®44/L WAF solution was used as the 100 mg KR®44/L test treatment solution.
- Differential loading: No
- Controls: Dilution water only
- Evidence of undissolved material: control, 0.95, and 3.1 mg KR®44/L test treatment solutions: clear and colourless, 9.8 mg KR®44/L test treatment: very slightly cloudy at initiation and at 24 hours, and clear and colorless with no visible particulates, surface film, undissolved test substance, or precipitate at 48 and 72 hours, 31 mg KR®44/L test treatment: slightly cloudy at initiation and at 24 hours, very slightly cloudy at 72 hours, 100 mg KR®44/L test treatment: were cloudy at initiation and at 24 hours, and slightly cloudy at 48 and 72 hours

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Source: obtained from the Culture Collection of Algae, University of Texas at Austin, USA, on 12 March 2019
- Age of inoculum: 2 days
- Method of cultivation: maintained in a temperature-controlled environmental chamber under continuous light. Periodically, new cultures were cloned from an existing culture derived from the parent stock.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.9 - 24.2 °C

pH:

7.4 - 9.5

Nominal and measured concentrations:

Nominal loading rates: 0 (control), 0.95, 3.1, 9.8, 31 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks with foam stoppers
- Type: closed
- Fill volume: 100 mL
- Aeration: No
- Initial cells density: 8.70 × 10^3 cells/mL cells/mL
- Control end cells density: 115 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP medium according to ASTM and OECD 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reagent water is produced by passing reverse-osmosis water through a series of deionization tanks, a laboratory water purification system consisting of carbon, demineralization, and organic adsorption cartridges, and then through a 0.2-µm filter. After preparation, the medium was filtered through 0.2-μm filters and, if needed, the pH of the medium was adjusted to 7.5 ± 0.1 using 0.1 N NaOH, if necessary.
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature: continuosly rom a flask containing deionized water using an electronic datalogger with thermistor probe, light intensity: daily (LI-COR Model LI-250A light meter), Temperature and pH: prior to distribution of the solutions to the test flasks (all parent soultions) and at 72 h: in composite solutions from all replicates of the control and each substance treatment (WTW Model pH 330i pH meter)

OTHER TEST CONDITIONS
- Photoperiod: continous light
- Light intensity and quality: 7.693 - 7.775 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscopic counting with a hemacytometer atest start and after 24, 48 and 72 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1 - 3.26
- Range finding study: yes
- Test concentrations: 0.010, 0.10, 1.0, 10, and 100 mg KR®44/L
- Results used to determine the conditions for the definitive study: The mean control cell density at termination was 93.0 × 104 cells/mL. The mean cell density at 72 hours was 88.0, 70.0, 64.4, 45.0, and 9.87 × 10^4 cells/mL in the 0.010, 0.10, 1.0, 10, and 100 mg KR®44/L treatments, respectively.

Reference substance (positive control):

yes

Remarks:

Zinc chloride

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: growth rate, yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

9.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: growth rate, yield

Effect concentrations 3

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

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

24.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% Cl: 20.0 - 28.2 mg/L

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: 9.8 mg KR®44/L test treatment: very slightly cloudy soultion at initiation, 31 mg KR®44/L test treatment: slightly cloudy soultion at initiation and at 24 hours, very slightly cloudy at 72 hours, 100 mg KR®44/L test treatment: were cloudy solution at initiation and at 24 hours, and slightly cloudy at 48 and 72 hours
- The statistically significant reduction in the 0.95 mg KR®44/L test treatment growth rate compared to the control was not considered biologically significant as the next highest level (3.1 mg KR®44/L) was not significantly reduced.

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- EC50: 0.17 mg/L (95% confidence limits: 0.17 - 0.18 mg/L)

Reported statistics and error estimates:

All statistical analyses were performed with SAS software (Version 9.3 for Windows). The NOEC values, based on growth rate and yield, were estimated using a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett’s test (p = 0.05) where the alternate hypothesis was the mean for the growth parameter was reduced in comparison to the control. Prior to the Dunnett’s test, a Shapiro-Wilk’s test and a Levene’s test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. If the results from the Shapiro-Wilk’s and Levene’s tests indicated normality and insignificant heterogeneity (i.e.,p > 0.01), the analysis was performed on the non-transformed raw data. In instances of nonnormality or heterogeneity (i.e., p < 0.01), a square root transformation was performed. If both
the non-transformed raw data and the transformed data exhibited non-normality or inequality of variance, a non-parametric analysis of variance was performed on the ranks of the raw data values. Non-parametric analyses were performed on all 24-hour data. Parametric analyses were performed on all 48- and 72-hour data.

Any other information on results incl. tables

Table1: Daily cell densities and % inhibition of the unicellular green alga, Pseudokirchneriella subcapitata, during a 72 -hour exposure to KEN-REACT®KR®44

 

Nominal Loading Rate Concentration (mg KR®44/L)	REP	Cell Density (cells/mL × 10^4)			Inhibition %
		24 Hours	48 Hours	72 Hours
Control	A	4.11	25.0	101
	B	4.22	25.7	118
	C	5.67	22.2	129
	D	5.56	27.4	106
	E	4.78	24.7	111
	F	5.00	27.7	126
	Mean	4.89	25.5	112	NA
0.95	A	3.00	19.3	84.0
	B	4.00	20.6	87.5
	C	4.33	23.4	93.5
	Mean	3.78	21.1^a	88.3^a	23
3.1	A	5.67	28.2	104
	B	4.00	23.0	103
	C	4.22	19.9	119
	Mean	4.63	23.7^*	109	6
9.8	A	4.56	19.4	82.5
	B	4.67	20.4	74.8
	C	4.33	24.1	77.8
	Mean	4.52	21.3^*	78.4^*	32
31	A	3.44	16.9	51.0
	B	3.44	17.4	54.8
	C	4.56	16.0	57.8
	Mean	3.81	16.8^*	54.5^*	53
100	A	1.78	6.33	10.3
	B	1.89	6.44	10.8
	C	2.22	5.67	14.2
	Mean	1.96^*	6.15^*	11.8^*	90

^aStatistically significant reduction not considered biologically significant as the next highest level was not significantly reduced.

* Significant reduction in cell density as compared to the control (one-tailed Dunnett’s test, p = 0.05).

NA = Not applicable.

Table 2: Daily replicate growth rates from test start an d% inhibition of the unicellular green alga, Pseudokirchneriella subcapitata, during a 72-hour exposure to KEN-REACT®KR®44

 

Nominal Loading Rate Concentration (mg KR®44/L)	REP	Mean Growth Rate (cells/mL/hour)			% Inhibition
		24 Hours	48 Hours	72 Hours
Control	A	0.0647	0.0700	0.0660
	B	0.0658	0.0705	0.0682
	C	0.0781	0.0675	0.0694
	D	0.0773	0.0719	0.0667
	E	0.0710	0.0697	0.0673
	F	0.0729	0.0721	0.0691
	Mean	0.0716	0.0703	0.0678	NA-
0.95	A	0.0516	0.0646	0.0635
	B	0.0636	0.0659	0.0640
	C	0.0669	0.0686	0.0650
	Mean	0.0607	0.0664	0.0642^a	5
3.1	A	0.0781	0.0725	0.0664
	B	0.0636	0.0682	0.0663
	C	0.0658	0.0652	0.0683
	Mean	0.0692	0.0686	0.0670	1
9.8	A	0.0690	0.0647	0.0632
	B	0.0700	0.0657	0.0619
	C	0.0669	0.0692	0.0624
	Mean	0.0686	0.0665^*	0.0625^*	8
31	A	0.0573	0.0618	0.0565
	B	0.0573	0.0624	0.0575
	C	0.0690	0.0607	0.0583
	Mean	0.0612	0.0616^*	0.0575^*	15
100	A	0.0298	0.0414	0.0343
	B	0.0323	0.0417	0.0350
	C	0.0390	0.0391	0.0388
	Mean	0.0337^*	0.0407V	0.0360V	47

^a Statistically significant reduction not considered biologically significant as the next highest level was not significantly reduced.

* Significant reduction in cell density as compared to the control (one-tailed Dunnett’s test, p = 0.05).

NA = Not applicable.

Table 3: Measured Concentrations of Titanium analytical marker during a 72 -hour growth inhibition toxicity test with unicellular green alga, Pseudokirchneriella subcapitata

 

Nominal KR®44 Test Substance Loading Rate (mg KR®44/L)	Measured Titanium Concentrationaas mg/L
	0 Hour	72 Hour
Control	<LODb	<LODb
0.95	<LODb	<LODb
3.1	<LODb	0.0200
9.8	<LODb,d	0.0239d
31	0.00756d	0.0428d
100	0.0217d	0.0258d
QC Fortification Spikes (% Recovery)
Low Spike (0.0100)c	0.0113 (113) 0.0101 (101)	0.0120 (120) 0.0121 (121)f
Mid Spike (0.100)c	0.118 (118)	0.125 (125)g
High Spike (10.0)c	8.94 (89)e 10.1 (101)	11.0 (110)e 12.0 (120)

a Measured Concentration (mg/L) = Measured concentration from curve (mg/L) × analysis volume (mL) / sample volume (mL).

b  Titanium LOD = 0.00300 mg titanium/L.

c  Nominal concentration in mg titanium/L.

d  Original result outside the standard curve. A less dilute aliquot analyzed and results reported.

e  Re-diluted in duplicate and re-analyzed, average of duplicate re-analyses reported.

f Re-analyzed in duplicate, results of duplicate re-analyses not reported, original analysis result reported.

g Re-diluted in duplicate and re-analyzed, average of original and duplicate re-analyses reported.

Table 4: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	131	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	< 35%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	2%	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.