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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted April 13, 2004, equivalent to the Commission Regulation (EC) No 440/2008, C.2., 2008

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The 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: 62.2, 33.2, 20.0, 9.63, 4.31, 2.20 and 0.94 mg test item/L,
- 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 of all test concentrations and the control were taken at the start of the test and at day 1. 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, duplicate samples from the aged test media of all test concentrations and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test 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.
- Analyses: 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 and 48 hours).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- 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 on the daphnids were observed. 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 2). After 24 hours 73 % of the nominal carbon content were found whereas 62 % were found after 48 hours stirring. This led to the decision of a stirring time of 24 hours for the third pre-experiment.
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. The effect on the daphnids was 20 % immobility at 10 and 100 mg test item/L.
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 (BlS-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 were excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the project number of the present study.
- Method: The test item is not well soluble in test medium and is therefore handled in accordance with OECD guidance document 23. To avoid physical effects of undissolved test item on the daphnids, no concentrations above the solubility limit of the test item in test water was tested. Therefore, one day prior to the introduction of the daphnids (= start of the test), a defined amount of the test item was added directly to the test water for each of the test concentrations of nominal 113.4, 51.5, 23.4 and 10.6 mg test item/L and was carefully stirred for 24 hours in the dark to dissolve as much of the test item as possible. The highest test item concentration of nominal 113.4 mg test item/L was prepared by mixing 119.2 mg test item into 1051.15 mL test water, for the test item concentration of nominal 51.5 mg test item/L, 54.2 mg test item were mixed into 1052.43 mL test water, for the concentration of nominal 23.4 mg test item/L, 24.6 mg were mixed into 1051.28 mL test water. The test item concentration of nominal 10.6 mg/L was prepared by mixing 26.5 mg test item into 2500 mL of 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 (WAF), was drawn off carefully and used as the test medium of the corresponding nominal test concentration. Furthermore, adequate volumes of the extracted phase of the test item concentration of nominal 10.6 mg/L were mixed into test water to obtain the test concentrations of nominal 4.80, 2.20 and 1.00 mg test item/L. The lowest concentrations (4.80, 2.20 and 1.00 mg/L) were not set up as WAFs but diluted by the next higher concentration, as it was technically not feasible regarding the initial weight.
One day prior to test medium renewal on Day 1, a defined amount of the test item was added directly to the test water for each of the test concentrations of nominal 113.4, 51.5 and 23.4 mg test item/L and was carefully stirred for 24 hours in the dark to dissolve as much of the test item as possible. The highest test item concentration of nominal 113.4 mg test item/L was prepared by mixing 119.1 mg test item into 1050.26 mL test water, for the test item concentration of nominal 51.5 mg test item/L, 54.1 mg test item were mixed into 1050.49 mL test water, for the concentration of nominal 23.4 mg test item/L, 24.6 mg were mixed into 1051.28 mL test water. The test item concentration of nominal 10.6 mg/L was prepared by mixing 26.5 mg test item into 2500 mL of 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. Adequate volumes of the extracted phase of the test item concentration of nominal 10.6 mg/L were mixed into test water to obtain the test concentrations of nominal 4.80, 2.20 and 1.00 mg test item/L.
The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
- Controls: In the control, test water was used without addition of the test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain/clone: clone 5
- Justification for species other than prescribed by test guideline: N/A
- Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.
- Breeding conditions: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicotus) freshly grown in the laboratories of ibacon.

ACCLIMATION
- Acclimation was not necessary since the test was performed in the same medium as the culturing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

21.1 to 21.4 °C in the freshly prepared media;
20.4 to 21.1°C in the aged test media

pH:

7.9 to 9.0 in the freshly prepared media;
7.8 to 8.6 in the aged test media; and thus, the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.5 to 8.9 mg/L in the freshly prepared media;
8.6 to 8.7 mg/L in the aged test media

Nominal and measured concentrations:

0, 0.94, 2.20, 4.31, 9.63, 20.0, 33.2, 62.2 mg test item/L (calculated concentrations based on TOC measurements)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers of approximately 110 mL volume containing as much test medium as possible (Le. the remaining head space was reduced to a technical possible minimum of some mL), kept closed during the whole period of the study with a conical glass stopper to avoid loss of the test item clue to volatilisation. For example, diisopropylamine as one of the hydrolysis products is a volatile compound.
- Type (delete if not applicable): closed
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Semi.static; no flow-through
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Medium: Elendt M4 Medium according to guideline
- Culture medium different from test medium: No
- Intervals of water quality measurement: 24 h

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark
- Light intensity: The light intensity was 290 to 610 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility - The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae).

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY - Yes
- Test concentrations: 0.1, 1, 10 and 100 mg test item/L and a control with 2 replicates per treatment group
- Results used to determine the conditions for the definitive study: Several Pre-experiments and range finding studies were collected, as the substance is difficult to test. The data of all pre-experiments together with the data 0f the study R 006/2017 led to the design of the main test. For details please refer to the section “Details on test solution”.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

22.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The measured concentrations are based on all hydrolysis products of the test item.

Remarks:

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:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.94 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The measured concentrations are based on all hydrolysis products of the test item.

Remarks:

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:

48 h

Dose descriptor:

LOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The measured concentrations are based on all hydrolysis products of the test item.

Remarks:

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:

- Behavioural abnormalities: No
- Observations on body length and weight: No
- Other biological observations: No
- Mortality of control: 0
- Other adverse effects control: No
- Abnormal responses: 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
- Relevant effect levels:
- Limit test: No
- Dose-response test: Yes
- EC50 = 1.02 mg/L (CI 95 %: 0.850 – 1.21)
- NOEC = 0.25 mg/L
- LOEC = 0.5 mg/L

Reported statistics and error estimates:

The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis. The NOEC and LOEC after 24 and 48 hours were calculated by Step-down Cochran Armitage test procedure.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Validity Criteria of the Study

-       Control Immobilisation Rate:           Was 0 % and furthermore no daphnid showed signs of disease or stress; thus, the validity criterion was met.

Dissolved Oxygen Concentration:            Was ≥ 8.6 mg O2/L in in all treatment groups at the end of the test; thus, validity criterion was met.

 

Analytical Results

All reported results refer to calculated concentrations corresponding to mean measured values since not all of the nominal test item concentrations of were within ± 20 % of the measured initial concentrations during the test (see table 1).

Table 1. Summary of Analytical Results           Sample Description Total Organic Carbon calculated concentration nominal concentration TOC [%] RSD   [mg test item/L] [mg Carbon/L] [mg Carbon/L]^1,2 of nominal [%] n [mg test item/L]               Control 0 n.a. n.a. n.a. 8 n.a. 1.0 0.6 0.56 94 23 8 0.94 2.2 1.32 1.32 100 25 8 2.20 4.8 2.87 2.57 90 7 7 4.31 10.6 6.34 5.76 91 3 8 9.63 23.4 13.99 11.95 85 8 8 20.0 51.5 30.78 19.83 64 18 8 33.2 113.4 67.79 37.18 55 4 8 62.2                 ^1mean value of all measured samples per treatment group ^2corrected by the mean of the control samples. 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) = 3.4 mg/L LOD: Limit of Detection (Inorganic Carbon) = 1.2 mg/L LOQ: Limit of Quantification = 1 mg Carbon/L

 

Biological Results    

Signs of Intoxication after 48 Hours:      After 48 hours of exposure no immobilisation of the test animals was observed in the control and the test item concentration of measured 0.94 mg/L and nominal 1.00 mg test item/L. A dose-related immobilisation of the test animals was observed from the test item concentration of measured and nominal 2.2 mg test item/L up to the test item concentration of measured 62.2 mg/L test item/L and nominal 113.4 mg test item/L. The derived effect concentrations are listed in table 2.

 

Table 2. Influence of 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite on the Mobility of Daphnia magna(data are presented on basis of measured values)
Endpoint [mg test item/L]	24 hours	48 hours
EC50	102	22.8
95 % CI	55.0 - > 113.4	14.8 - 41.1
EC20	29.7	5.02
95 % CI	17.8 -54.7	2.43 - 7.95
EC10	15.6	2.28
95 % CI	6.33 -24.9	< 1.0 -4.10
NOEC	9.630	0.940
LOEC	20.000	2.200
Values refer to calculated test concentrations CI: Confidence interval n.d.: not determinable NOEC and LOEC were determined by Step-down Cochran-Armitage Test.

Further results on the mobility of D. magna, test conditions, and results of the preliminary tests are presented below in table 3 to 8.

Validity criteria fulfilled:

yes

Conclusions:

In an acute toxicity test according to the OECD guideline 202 with Daphnia magna an EC50 of 22.8 mg/L was determined for 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite.

Executive summary:

The 48-hr-acute toxicity of at 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite to Daphnia magna was studied under semi-static conditions. Test species were exposed to control, and test chemical at measured concentrations of 0.94, 2.20, 4.31, 9.63, 20.0, 33.2, and 62.2 mg a.i./L for 48 hr. 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.

Immobilisation and sublethal effects were observed daily. The 48-hour EC₅₀ was 22.8 mg a.i./L.  The 48-hr NOEC based on immobilization was 0.940 mg a.i./L.  

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

 

Results Synopsis

 

Test Organism Age (e.g. 1^stinstar): ≤ 24 h

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

 

NOEC: 0.940 mg/L

LOEC: 2.2 mg/L

EC₅₀: 22.8 mg/L       95% C.I.: 14.8 to 41.1 mg a.i./L

Endpoint(s) Effected: Mobility

 

Description of key information

In an acute toxicity test according to the OECD guideline 202 with Daphnia magna an EC₅₀ of 22.8 mg/L was determined for 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

22.8 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.