3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

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:

2018-07-02 to 2018-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted March 23, 2006, corrected July 28, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item hydrolyses fast in water and acetonitrile (half-life = 0.5 h, 22 °C); according to study R 006/2017; 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (BIS- Amidite): Abiotic degradation – Hydrolysis – Pre-Test; WeylChem lnnoTec GmbH; see section 4.8 for details.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the carbon content of the test item 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite were analysed in the duplicate test media samples from all test concentrations and in the duplicate control samples from all sampling times (0, 24, 48 and 72 hours).
- Sampling method: The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated during test period under the same conditions as in the actual test and from these test media duplicate samples were taken after 24 and 48 hours. The collecting of samples after 24 and 48 hours from the actual test itself was not possible, since the test media volumes in the test were too small for the analytical requirements. Duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
- Sample storage conditions before analysis: All samples were stored in a refrigerator (4 ± 4 °C), protected from light until analysis was performed. Afterwards the samples were again stored refrigerated and will be kept stored up to the date of the final report.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Defined amounts of the test item were added directly to the test water for each test concentration and were carefully stirred for 48 hours in the dark to dissolve as much of the test item as possible. The highest test item concentration of 100 mg test item/L was prepared by mixing 200.0 mg test item into 2000 mL test water, for the test item concentration of 32 mg test item/L, 64.0 mg test item were mixed into 2000 mL test water, for the concentration of 10 mg test item/L, 20.0 mg were mixed into 2000 mL test water. The concentration of 3.2 mg test item/L was prepared by mixing 16 mg into 5000 mL test water and for 1.0 mg test item/L, 11 mg were mixed into 11000 mL test water. After cessation of mixing and a following period (1 hour) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration. Each concentration was prepared separately. The test media were prepared just before introduction of the algae (= start of the test).
- Controls: In the control, test water was used without addition of the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): no vehicle used

PRE-EXPERIMENTS: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
A first pre-experiment to determine the solubility of the test item showed that the test substance was not completely solubilised by stirring overnight in a concentration of 100 mg test item/L. The carbon content of this solution was about 76 % of the nominal content.
A second pre-experiment with stirring of 3 hours (6x half-life time, as suggested by OECD TGD 23) led to recoveries of about 10% carbon content in the test concentration of 100 mg test item/L. The experiment was run with concentrations of 0.1, 1, 10 and 100 mg test item/L and a control with 2 replicates per treatment group. No effects were observed up to and including the concentration of 10 mg test item/L. At 100 mg test item/L the inhibition on growth rate and yield was 57.0% and 15.7%. The concentration was considered to be too low. Therefore, it was decided to proceed with an additional solubility pre-test. In this solubility pre-test 100 mg test item/L were stirred for 24 and 48 hours. The samples were drawn of the water phase after separation of undissolved test item. These samples were analysed via measurement of Total Organic Carbon (reasoning of choice of analytical method is presented in the appendix II.2). After 24 hours 59 % of the nominal carbon content were found whereas 69 % were found after 48 hours stirring. The third pre-experiment led to recoveries of about 50% carbon content in the test concentration of 100 mg test item/L and 100 % carbon content in the test concentration of 10 mg test item/L. The experiment was run with concentrations of 0.1, 1, 10 and 100 mg test item/L and a control with 2 replicates per treatment group. No effects were observed up to and including the concentration of 1 mg test item/L. At 10 mg test item/L the inhibition on growth rate and yield was 27.2 % and 6.1 %, respectively, at 100 mg test item/L the inhibition on growth rate and yield was 99.4 % and 100 %.
Testing of the hydrolysis product was not conducted as the test item hydrolyses very quickly and even the first hydrolysis product hydrolyses further to smaller hydrolysis products which also hydrolyse. The composition of the hydrolysis products is continuously changing and therefore it is not possible to focus on one single hydrolysis product. For further details please refer to study R 006/2017; 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (BIS-Amidite): Abiotic degradation – Hydrolysis – Pre-Test; WeylChem lnnoTec GmbH. The data of all pre-experiments together with the data of the study R 006/2017 led to the design of the main test. The pre-experiments were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study number of the present study.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Origin: The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
- Reference Item: For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

ACCLIMATION
- Acclimation period: No acclimation was necessary
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22.5 to 23.1 °C

pH:

8.1 to 8.6 at test start and
8.7 to 9.8 at test end
(The pH in the control increased by more than 1.5 units medium.
However, this does not invalidate the tests since the validity criterion was met.)

Nominal and measured concentrations:

Due to the limit of water solubility being less than the nominal concentration of the test item Water accommodated Fractions (WAF) were prepared in accordance with OECD guidance document 23. Nominal loading rates of 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.2) and a control were tested. In order to facilitate the readability of the text it is referred in the present document to mg test item/L, implying the hydrolysis products of the test item as well.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: All reported results refer to nominal values since the concentrations of carbon content of the test item were within ± 20 % of the nominal, quantifiable concentrations during the test

Remarks:

The measured concentrations are based on all hydrolysis products of the test item. The test item hydrolyses very quickly, and the hydrolysis products react further with water to smaller hydrolysis products which also hydrolyse. Hence, it was refrained from analysing one hydrolysis product, as no major and stable hydrolysis product could be found. To comprise all hydrolysis products of the test substance, TOC is the best suitable method for the present study.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: All reported results refer to nominal values since the concentrations of carbon content of the test item were within ± 20 % of the nominal, quantifiable concentrations during the test

Remarks:

The measured concentrations are based on all hydrolysis products of the test item. The test item hydrolyses very quickly, and the hydrolysis products react further with water to smaller hydrolysis products which also hydrolyse. Hence, it was refrained from analysing one hydrolysis product, as no major and stable hydrolysis product could be found. To comprise all hydrolysis products of the test substance, TOC is the best suitable method for the present study.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: All reported results refer to nominal values since the concentrations of carbon content of the test item were within ± 20 % of the nominal, quantifiable concentrations during the test

Remarks:

The measured concentrations are based on all hydrolysis products of the test item. The test item hydrolyses very quickly, and the hydrolysis products react further with water to smaller hydrolysis products which also hydrolyse. Hence, it was refrained from analysing one hydrolysis product, as no major and stable hydrolysis product could be found. To comprise all hydrolysis products of the test substance, TOC is the best suitable method for the present study.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: All reported results refer to nominal values since the concentrations of carbon content of the test item were within ± 20 % of the nominal, quantifiable concentrations during the test

Remarks:

The measured concentrations are based on all hydrolysis products of the test item. The test item hydrolyses very quickly, and the hydrolysis products react further with water to smaller hydrolysis products which also hydrolyse. Hence, it was refrained from analysing one hydrolysis product, as no major and stable hydrolysis product could be found. To comprise all hydrolysis products of the test substance, TOC is the best suitable method for the present study.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: All reported results refer to nominal values since the concentrations of carbon content of the test item were within ± 20 % of the nominal, quantifiable concentrations during the test

Remarks:

The measured concentrations are based on all hydrolysis products of the test item. The test item hydrolyses very quickly, and the hydrolysis products react further with water to smaller hydrolysis products which also hydrolyse. Hence, it was refrained from analysing one hydrolysis product, as no major and stable hydrolysis product could be found. To comprise all hydrolysis products of the test substance, TOC is the best suitable method for the present study.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.963 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by three parametric normal concentration distribution function (CDF). For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Bonferroni-Welch t-test (yield) and Williams t-test (growth rate). The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat® Solutions GmbH.

Validity Criteria of the Study

Cell Density Increase in Control Cultures: 254.0-fold increase within 72 hours and thus, the validity criterion was met.

 

Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 20.2 % and thus, the validity criterion was met.

 

Coefficient of Variation of Average Growth between Control Replicates: 0.4 % and thus, the validity criterion was met.

 

Biological Results

Growth Inhibition: The 72-hour EyC50 was calculated to be 8.59 mg test item/L and the ErC50 20.4 mg test item/L. The 72-hour EyC10 was calculated to be 3.96 mg test item/L and the ErC10 8.11 mg test item/L. The 72-hour NOEyC was determined to be 3.2 mg test item/L and the associated 72-hour LOEyC of 10 mg test item/L. The 72-hour NOErC was determined to be 3.2 mg test item/L and the associated 72-hour LOErC is 10 mg test item/L. For further details see Table 1.

Table 1. Influence of 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite on the Growth of Pseudokirchneriella subcapitata

Parameter	Yield [mg test item/L]	Growth rate [mg test item/L]
72-hour EC50	8.59	20.4
95 % conf. interval	8.09 - 9.12	20.0 - 20.8
72-hour EC20	5.17	11.1
95 % conf. interval	4.64 - 5.74	10.8 - 11.5
72-hour EC10	3.96	8.11
95 % conf. interval	3.41 - 4.59	7.81 - 8.40
72-hour NOEC	3.2	3.2
72-hour LOEC	10	10
n.d. = not determinable Values refer to nominal test concentrations

 

Microscopic Examination: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

 

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of carbon content of the test item were within ± 20 % of the nominal, quantifiable concentrations during the test (see Table 2).  Only the nominal concentration of 1 mg test item /L corresponded to only 40 mg test item/L on basis of measured values. This is of minor importance as in 3.2 mg/L no effect was observed.

 

Table 2. Summary of Analytical Results

Sample Description		Total Organic Carbon				Calculated
nominal concentration		TOC	[%]	RSD		Concentration
[mg test item/L]	[mg Carbon/L]	[mg Carbon/L]^1	of nominal	[%]	n	[ mg test item/L]
Control	0	2.54	n.a.	n.a.	8	n. a.
1.0	0.60	0.24	40	172	8	0.40
3.2	1.91	1.88	98	13	8	3.1
10	5.98	6.04	101	11	8	10.1
32	19.13	18.69	98	2	8	31.3
100	59.77	58.95	99	1	8	99

¹mean value of all measured samples per treatment group
RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable
italics: values below LOQ
LOD: Limit of Detection (Total Carbon) = 2.5 mg/L
LOD: Limit of Detection (Inorganic Carbon) = 0.9 mg/L
LOQ: Limit of Quantification = 5 mg Carbon/L

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC50 based on growth rate of the test item in Pseudokirchneriella subcapitata is 20.4 mg/L (95 % CL 20.0 to 20.8 mg/L).

All reported results refer to nominal values since the concentrations of carbon content of the test item were within ± 20 % of the nominal, quantifiable concentrations during the test

Executive summary:

In a 72-hour algae growth inhibition test cultures of Pseudokirchneriella subcapitata were exposed to 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite at nominal concentrations of 0, 1.0, 3.2, 10, 32, 100 mg test item/L under static conditions in accordance with the OECD 201 (2006) and GLP. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of carbon content of the test item were within ± 20 % of the nominal, quantifiable concentrations during the test. 

The NOErC and ErC₅₀values based on the growth rate were 3.2 and 20.4 mg a.i./L, respectively.  The % growth inhibition in the treated algal culture as compared to the control ranged from -0.7 to 100 % (see table 7 above).

There were no compound related phytotoxic effects.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae growth inhibition study according to OECD guideline 201.

Results Synopsis

 

Test Organism: Pseudokircheriella subcapitata

Test Type (Flowthrough, Static, Static Renewal): static

 

Parameter	Yield [mg test item/L]	Growth rate [mg test item/L]
72-hour EC50	8.59	20.4
95 % conf. interval	8.09 - 9.12	20.0 - 20.8
72-hour EC20	5.17	11.1
95 % conf. interval	4.64 - 5.74	10.8 - 11.5
72-hour EC10	3.96	8.11
95 % conf. interval	3.41 - 4.59	7.81 - 8.40
72-hour NOEC	3.2	3.2
72-hour LOEC	10	10
n.d. = not determinable Values refer to nominal test concentrations *In order to facilitate the readability of the text it is referred in the present document to mg test item/L, implying the hydrolysis products of the test item as well.

Endpoint(s) Effected:  Growth rate and Yield

Description of key information

In a 72-hour algae growth inhibition test cultures of Pseudokirchneriella subcapitata were exposed to 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite at nominal concentrations of 0, 1.0, 3.2, 10, 32, 100 mg test item/L under static conditions in accordance with the OECD 201 (2006) and GLP.

The following effect levels - based on the growth rate - are calculated:

- 72 h ErC50: 20.4 mg/L (95 % C.L. 20.0 to 20.8 mg/L);

- 72 h NOErC: 3.2 mg/L

- 72 h LOErC: 10 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

20.2 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L

Additional information

The test item hydrolyses very quickly and even the first hydrolysis product hydrolyses further to smaller hydrolysis products which also hydrolyse. The composition of the hydrolysis products is continuously changing and therefore it is not possible to focus on one single hydrolysis product for toxicity testing. In addition, no major and stable hydrolysis product could be found. Therefore, the measured test concentrations imply the test item and all its hydrolysis products.