Hydrogen tetrakis[2,2-bis[(allyloxy)methyl]butan-1-olato-O1]bis(ditridecyl phosphito -O'')titanate(2-)

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:

28 - 31 Jan 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:

2006

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen tetrakis[2,2-bis[(allyloxy)methyl]butan-1-olato-O1]bis(ditridecyl phosphito -O'')titanate(2-)

EC Number:

264-709-9

EC Name:

Hydrogen tetrakis[2,2-bis[(allyloxy)methyl]butan-1-olato-O1]bis(ditridecyl phosphito -O'')titanate(2-)

Cas Number:

64157-14-8

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

dihydrogen 2-{[(di{[bis(tridecyloxy)phosphanyl]oxido}tris({2,2-bis[(prop-2-en-1-yloxy)methyl]butyl}oxido)titaniotetrakis(ylium))oxido]methyl}-1-(prop-2-en-1-yloxy)-2-[(prop-2-en-1-yloxy)methyl]butane

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

LC-MS/MS (for reference substance Trimethylolpropane diallyl ether), ICPMS (for reference substance Titanium )

Details on sampling:

- Concentrations: 0 (control), 0.95, 0.95 (abiotic), 3.1, 9.8, 31.0, 100 mg/L
- Sampling method: The concentration of TMPDAE was measured in test solution samples collected at initiation (0 hour) and termination (72 hours) of the definitive test.
- Sampling method: The concentration of titanium was measured in test solution samples collected at initiation (0 hour) of the definitive test. Time 0 hour samples were collected from parent solutions.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The five test solutions were prepared as water accommodated fractions (WAF). On 27 January 2019, 0.1001 g of KR®55 was weighed into a plastic weighing funnel and dropped into a glass carboy containing 1.0 L of test medium to prepare a 100 mg/L primary solution. A magnetic stir bar was added to the glass jar. The solution was stirred, at room temperature and protected from light, for approximately 23 hours with a vortex no greater than approximately 25% of the solution depth. After stirring, the solution was removed from the stir plate and allowed to settle for approximately 1 hour. The solution appeared clear and colorless. After the hour settling, approximately 200 mL of the solution was decanted and discarded. Appropriate aliquots of the primary solution were diluted to 0.50 L with test medium to prepare the 0.95, 3.1, 9.8, and 31 mg/L test treatment solutions. The primary solution was used as the 100 mg/L test treatment solution.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source: The parent stock was obtained from the Culture Collection of Algae, University of Texas at Austin, USA on 21 August 2018. The prepared cultures were maintained in a temperature-controlled environmental chamber under continuous light. Periodically, new cultures were cloned from an existing culture derived from the parent stock. All cultures were maintained under the same conditions as those used for testing.
- Age of inoculum (at test initiation): The algal culture used for this test was three days old and the biomass had increased exponentially (i.e., specific growth rate of 1.4 day-1) during the culture period.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.3-24.1°C

pH:

7.2-7.8

Nominal and measured concentrations:

Nominal loading rates: 0 (control), 0.95, 3.1, 9.8, 31, 100 mg/L
Mean measured TMPDAE concentrations: < LOD (control), 0.153, 0.426, 1.57, 4.23, 15.8 mg/L
Mean measured titanium concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flask
- Fill volume: 100 mL
- Initial cells density: 0.667 × 10^4 cells/mL
- Control end cells density: 70.2 ^ 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: A continuous recording of environmental chamber temperature was made from surrogate flask containing water using an electronic datalogger with thermistor probe. Temperature and pH were measured in all parent solutions prior to distribution of the solutions to the test flasks. At 72 hours, temperature and pH were measured in composite solutions from replicates A, B, C, D, E, and F in the control and from replicates A, B, and C in each test substance treatment. All temperature and pH measurements of the test solutions were performed with a WTW Model pH 330i pH meter.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 8,059 to 8,102 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscopic counting with a hemacytometer at 24, 48, and 72 hours (±1 hour)


TEST CONCENTRATIONS
- Range finding study: no

Reference substance (positive control):

yes

Remarks:

zinc chloride

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

59 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%-CI: 41.5 - 76.6 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%-CI: > 100 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

85.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%-CI: 77.3 -93.2 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

28.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95%-CI: 20.9 - 37.0 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

69.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95%-CI: 63.4 - 75.8 mg/L

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95%-CI: 31.8 - 48.2 mg/L

Details on results:

- Exponential growth in the control: yes
-Observation of abnormalities: no

Results with reference substance (positive control):

- Results with reference substance valid? Results were consistent with previous testing conducted in 2018.
- EC50: 0.17 mg/L (95%-CI: 0.17 - 0.18)

Any other information on results incl. tables

BIOLOGICAL RESULTS:

Percent inhibition in algal growth at 72 hours, as compared to the control, ranged from -19% at the concentration of 9.8 mg/L to 71% at the concentrations of 100 mg/L.

Table 1: Inhibitions of Growth rate and Yield after 72 h incubation. 

Nominal test substance loading rates [mg/L]	Growth rate (mean) [cells/mL/hour]	Inhibition of growth rate [%]	Yield (mean) [cells/mL × 10^4]	Inhibition of yield [%]
Control	0.0646		69.5
0.95	0.0662	-2	78.1	-12
3.1	0.0645	0	68.9	1
9.8	0.0669	-4	82.6	-19
31	0.0629	3	61.2	12
100*	0.0477	26	20.0 *	71

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

ANALYTICAL RESULTS:

TMPDAE:

The mean measured concentrations of TMPDAE were between 13.6 to 16.1% of the nominal concentrations.

Table 2: The measured concentrations of measured concentrations of TMPDAE during a 72‑Hour Growth Inhibition Toxicity Test with Unicellular Green Alga,Pseudokirchneriella subcapitata,to KR^®55

Nominal KR^®55 Test Substance Loading Rate (mg/L)	Measured TMPDAE Concentration^aas mg/L
	0 Hour	72 Hours	Mean Measured
0 (control)	<LOD	<LOD	<LOD
0.95	0.149	0.156	0.153
0.95 Abiotic	NA	0.154	0.152*
3.1	0.331	0.521	0.426
9.8	1.56	1.57	1.57
31	3.41	5.06	4.23
100	15.6	16.0	15.8

* = 0.95 mg/L value at 0 hour used for 0.95 mg/L abiotic value at 0 hour for calculation.

NA = Not applicable.

LOD = 0.000581 mg/L.

Titanium:

Table 3: The measured concentrations of measured concentrations of titanium during a 72‑Hour Growth Inhibition Toxicity Test with Unicellular Green Alga,Pseudokirchneriella subcapitata,to KR^®55

Nominal KR^®55 Test Substance Loading Rate (mg/L)	Measured Titanium Concentration^aas mg/L 0 Hour
0 (control)	<LOD
0.95	<LOD
3.1	<LOD
9.8	<LOD
31	<LOD
100	<LOD

a = Measured Concentration (mg/L) = Measured concentration from curve (ng/mL) × analysis volume (mL) / sample volume (mL) / 1,000 (ng/L / mg/mL).

LOD = 0.00300 mg/L.

VALIDITY CRITERIA:

The test acceptability criteria in the OECD Test Guideline 201 were met for this study. The number of algal cells in the control at test termination was greater than 16 times the number initially inoculated which verified logarithmic phase growth. The overall mean coefficient of variation for daily growth rates in the control replicates during the course of the test did not exceed 35%. The coefficient of variation of average specific growth rates during the whole test period in control replicates did not exceed 7%. The pH in the control did not increase more than 1.5 units during the study.

Table 4: Validity criteria for OECD 201

Validity Criterion	Required	This study
Increase of the cell growth in the control cultures	Exponentially, ≥ 16-fold	105-fold
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control culture	≤ 35%	10%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	2%

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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