N-methyl-P,P-bis(2-methylaziridin-1-yl)phosphinamide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study is performed according to the OECD 202 guideline.

Justification for type of information:

Study on-going

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 1000010405
- Manufacturing date: 15 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Fridge (4±3°C)

Analytical monitoring:

yes

Details on sampling:

- Frequency of sampling: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).
- Number of samples: Sampling will consist of duplicate samples taken per treatment.
- Analytical monitoring: Concentration of dissolved organic material in the WAFs will be checked by analysis of total organic carbon (TOC) in the control medium and the test loading rates. TOC analysis will not be performed in compliance with OECD GLP principles but will be adapted to fit the specific parameters of the test item, in accordance with ISO 17025.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The study will be carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) will be prepared under closed conditions and by slow-stirring.
The mixing vessels will be 1 L cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. A magnetic stirring bar will be placed in each mixing vessel completely filled with test water (with a minimum headspace). The loading rates of the test item will be weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and will be immediately sealed with screw caps after weighing. Each glass flask will be placed in a water bath for 10-15 minutes at approx. 50°C, followed by sonication for approx. 10 minutes. Then the mixing vessels will be carefully filled with the contents of the glass flasks and thereafter will be closed immediately. The mixing will be initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 +/- 2 hours of gentle stirring in the dark at room temperature, the WAFs will be allowed to stand for at least 1 hour before use. The first 100 mL will be discarded via the drain port. Then the WAFs will be directly added into test vessels (without headspace) or will be filtered if necessary. After filling and introduction of daphnids the test vessels will be sealed immediately. The test will be carried out without adjustment of the pH.
Depending on the behaviour of the test substance in test water, minor changes in the method of preparation may be carried out.

Controls: Test water without test substance but treated in the same way as the test substance solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Species: Daphnia magna (Straus), clone 5
- Sex: Female
- Origin: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at test start: < 24 hours old
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Breeding conditions: Daphnids are cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids is performed in all-glass vessel containing test water. Cultures are maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids are fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water is changed at least once per week. These culture conditions allow to maintain the daphnids in the parthenogenetic reproductive stage.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

No data

Hardness:

A water hardness between 140 and 250 mg.L-1 (as CaCO3) is recommended.

Test temperature:

Controlled environment cabinet (20°C ± 2°C); should not vary by more than 1°C at the end of the test)

pH:

Between 6.0-9.0. Should not vary by more than 1.5 units at the end of the test in any solution.

Dissolved oxygen:

Dissolved oxygen concentration at the end of the test should be ≥ 60% of the air-saturation value in controls and test vessels.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Range-finding test on-going

Details on test conditions:

TEST PROCEDURE AND CONDITIONS
- Test duration: 48 hours
- Test type: Semi-static, in order to prevent the dissipation of the test item from test solutions because of its volatility and the expected degradation of some constituents.
- Test vessels: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel will be uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Number of daphnids: 20 per treatment group (control and loading rate of 100 mg.L-1), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace
- Number of replicates: 4 replicates with daphnids per treatment group.
- Aeration: No aeration of the test solutions
- Feeding: No feeding
- Introduction of daphnids: Daphnids will be introduced into the test vessel each completely filled with test solution and without headspace as soon as possible after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Test water: Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED
Immobility: Immobility and abnormal behaviour will be determined by visual observation after 24 and 48 hours. Immobile animals will be eliminated from the vessels as soon as they will be discovered. Daphnids are considered to be immobile if they are not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all treatment group.
- Temperature of medium: Measured continuously in a “temperature control” vessel next to the test vessels, over the study period, beginning at the start of the test.

DATA HANDLING
- Defining exposure concentrations: Effective concentrations will be determined considering nominal WAF concentrations (nominal loading rate values).
- Estimation of EL50 after 24 and 48 hours of exposure (if applicable): The data will be analysed by regression to determine the EL50 including the 95 % confidence interval by using the computer program ToxRat. The highest loading rate without effect and lowest concentration with 100% immobilisation will be determined by visual observation. Optionally, other statistical analyses may be performed if appropriate.

RANGE FINDING TEST
- Preparation of test solutions: The range-finding test is carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rates of 1, 10, 32 and 100 mg.L-1 and to a control. Two method of preparation for the WAFs is tried: heating/sonication and solvent conditions. The results of this preliminary test will be used in order to determine the range of loading rates to be tested in the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

preliminary study

Details on results:

Preliminary study: no sign of stress was observed on the mobile daphnids after 48 hours of exposition.

Results with reference substance (positive control):

No results available. Study on-going.

Reported statistics and error estimates:

No results available. Study on-going.

Validity criteria fulfilled:

not applicable

Conclusions:

Preliminary study: no sign of stress was observed on the mobile daphnids after 48 hours of exposition.

Executive summary:

A study is performed under GLP conditions to assess the acute toxicity of test item to Daphnia magna. The method followed is designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended byCommission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

The daphnids are exposed to Water Accommodated Fractions (WAFs) of the test item. The immobility of the daphnids is determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the control and the WAFs is checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

No sign of stress was observed on the mobile daphnids after 48 hours of exposure in the preliminary study.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2018-10-01 to 2018-10-09

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)

Deviations:

no

Principles of method if other than guideline:

Not relevant.

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed in November 2017

Specific details on test material used for the study:

FCBA reference: 1711228F11
Batch number:1 00001 0405
Manufacturing date: June 15,2017
CAS number: No.85068-72-0
Nominal content of the active substance: 97.5 % wlw
Manufacturer: ARIANEGROUP SAS
Test item physical state: Solid whitish cast
Test item quantity received at test facility: 300 g
Reception date: November 06,2017
Storage conditions: Fridge
Stability : re-inspection date: May 02,2019

Analytical monitoring:

yes

Details on sampling:

ln order to verify the nominal or initial concentrations of the test item and maintenance of the concentrations during the ecotoxicological testing, chemical analysis of the test item in daphnids test solutions were performed according to the Analytical protocol FCBA Chimie No.411-e version 2: Method for the chemical analysis of Total Carbon (TC) in samples contained in the test item METHYL BAPO from daphnids ecotoxicology solutions by a Total Organic Carbon Analyzer following protocol:
- At the beginning of the test (T = 0h)
- At the end of the test (T= 48h)

Schedule analysis at initial time (T0h):
T0h Control
T0h 55 mg/L
T0h 324 mg/L
T0h 1050 mg/L
All the solutions were analysed taken directly from the starting solutions without daphnids. Schedule analysis at final time (T48h):
T48h Control
T48h 55 mg/L
T48h 324 mglL
T48h 1050 mg/L
One replicate was used for chemical analyses.
The measured concentrations of Total Carbon (TC) in samples did not vary more than ±20 % of nominal or initial measured concentration during the test.
All the solutions were kept in refrigerator or freeze in order to be analyzed if necessary or relevant.

Vehicle:

yes

Remarks:

WAF

Details on test solutions:

Preparation of test solutions for the testing
A special solubilisation protocol was canied out before the experimentation, according to the OECD n'23 guidance document on aquatic toxicity testing of difficult substances and mixtures.
This method consisted in stirring each test item nominal concentrations in test medium during 24 hours, in the dark and 20± 2°C. (the WAF was prepared by adding directly the test item in flasks containing test medium).
Six concentrations (determined from a non-GLP preliminary test, in a geometric serie, were performed plus a Control (Test medium).
Based on the results of the preliminary test, the following test concentrations 55 - 100 - 180 - 324 - 583 and 1050 mg test item /L were proposed for the definitive test.

The test solutions plus Control (test medium) were then filtered hrough a membrane filter (cellulose acetate 0.45 µm) to remove suspended particles: solutions obtained are named WAF (Water Accommodated Fraction).

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Sfraus, clone number 5, from at least the third generation (l.e. the third brood) obtained with a non-cyclic parthenogenesis reproduction.
The daphnids breeding was carried out in closed boftles placed in a climatic chamber free from any toxic vapour and in medium M4.
Daphnids used in this experimentation were previously filtered in order to obtain animals from a size between 560 pm and 800 pm (corresponding to daphnids less than 24 hours old).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

250 mg/L of CaCO3

Test temperature:

19.8 - 20.0 °C

pH:

8.1

Dissolved oxygen:

9.1 mg/L

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal concentrations: 55 - 100 - 180 - 324-583 and 1050 mg/L (loading rates)

Details on test conditions:

The test contained a minimum of 28 glass test tubes as follow:
- Control: 4 replicates
- Concentration 55 mg/L: 4 replicates
- Concentration 100 mg/L:4 replicates
- Concentration 180 mglL:4 replicates
- Concentration 324 mg/L: 4 replicates
- Concentration 583 mglL: 4 replicates
- Concentration 1050 mg/L: 4 replicates
The pH of the solutions did not remain in the range 6 - 9, no pH adjustment was made with HCI and NaOH.
Each glass test tube contained approximately 10 mL of each test solution and 5 daphnids they were covered in order to avoid evaporation and dust deposition.
The test tubes were pooled for chemical analyses at the end of the test.

lncubation
The test was performed in the dark between 18 and 22"C bul constant within t1 "C. Daphnids were not fed during the test period.
Observations of the daphnid's immobility and behaviour were done after 24 and 48 hours in control and exposed groups.
Any sign of stress observed on the daphnids after 48 hours of exposure to the test item were reported.
pH and dissolved oxygen were measured at the beginning and at the end of the test for each test solutions (including controls).

TEST MEDIUM:
The medium was prepared from concentrated stock solutions in ultrapure water as described in the guideline

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Light intensity and quality: Darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilisation

RANGE-FINDING STUDY
- Range finding study
- Test concentrations:
WAF Control
WAF 1 mg/L
WAF 10 mg/L
WAF 100 mg/L
- Results used to determine the conditions for the definitive study: No effect observed at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

372.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Loading rates

Remarks:

[308.2-451.8]

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

139.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Loading rates

Remarks:

[116.3-166.0]

Details on results:

Validity criteria for the test:
- lmmobilised daphnids in the Control: Criteria for OECD 202: < 10%, validity criteria conformity: Yes (0%)
- Dissolved Oxygen at the end of the test: Criteria for OECD 202: > 3 mg/L, validity criteria conformity: Yes (8.6 mg/L)

Results with reference substance (positive control):

The last toxic response obtained on the reference item K2Cr2O2 (September 18, 2018) gives an ECso-24h of 1.01 mg/L (0.91 - 1.10), indicating that the daphnids sensitivity is correct (within the accepted interval of 0.6 - 2.1 mglL fixed by the lnternational Standard ISO 6341 December 2012 as mentioned in the OECD 202 Guideline) and in accordance with historical data obtained by the laboratory.

Reported statistics and error estimates:

The raw data can permit to calculate the ECso with the softvvare Minitab 17.2.4 with Probits analysis.

Daphnids immobilisation

Raw data (daphnids immobilisation in cumulative number for each observation time 24 and 48 hours) are presented below.

Nominal test item concentration (mg/L)	Number of daphnids exposed at the start	lmmobilised daphnids (%)
		at 24h	at 48h
Control	20	0 (0%)	0 (0%)
55	20	0 (0%)	0 (0%)
100	20	1 (5%)	5 (25%)
180	20	0 (0%)	15 (75%)
324	20	6 (30%)	19 (95%)
583	20	19 (95%)	20 (100%)
1050	20	19 (95%)	20 (100%)

pH and dissolved oxygen values during the final test

Nominal test item concentration (mg/L)	pH		Dissolved O2 (mq/L)
	T0h	T48h	T0h	T48h
Control	8.1	7.5	9	8.8
55	8.1	7.6	8.9	8.8
100	8.1	7.6	8.9	8.8
180	8.1	7.5	8.9	8.7
324	8.1	7.6	9.1	8.7
583	8.1	7.6	9	8.6
1050	8.2	7.6	8.9	8.7

Chemical analysis of algal solutions

The results of chemical analyses of samples at initial measurement and after 48 hours of exposure are presented in the following tables:

Nominal test item loading rate	Sample lD	Concentration (mg TC/L)	Dilution	Concentration (mg TC/L)
Control Abiotic T0h	T0h Control	< 5	/	< 5
55 mg/L Abiotic T0h	T0h 55 mg/L	24.65	/	24.65
324 mg/L Abiotic T0h	T0h 324 mg/L	29.02	5	145.1
1050 mg/L Abiotic T0h	T0h 1050 mg/L	47.57	10	475.7
Nominal test item loading rate	Sample lD	Concentration (mg TC/L)	Dilution	Concentration (mg TC/L)
Control Abiotic T48h	T48h Control	< 5	/	< 5
55 mg/L Abiotic T48h	T48h 55 mg/L	25.77	/	25.77
324 mg/L Abiotic T48h	T48h 324 mg/L	30.1	5	150.5
1050 mg/L Abiotic T48h	T48h 1050 mg/L	48.99	10	489.9

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item METHYL BAPO on the toxicological endpoint "immobilisation" give an EC50-24h= 372.3 mg/L [308.2 - 451.8] and an EC50-48h = 139.4 mg/L [116.3 - 166.01 based on nominal test item concentrations.

Executive summary:

The aim of this study was to assess the effects of the test item METHYL BAPO on the daphnids Daphnia magna, according to the OECD 202 guideline, in static conditions.

This study was carried out in accordance with the study planno.17/1228F/f dated September 12,2018.

There was no deviation and no amendment to the study plan during this study.

The concentrations which immobilise 50% of the daphnids exposed to the test item after 24 and 48 hours, reported as EC50 -24h and EC50-48h were evaluated, relative to the tested concentrations.

Chemical analyses of the test item in the test solutions was performed by a Total Organic Carbon (TOC) analyzer in order to check the test item stability throughout the study.

The results obtained for the test item METHYL BAPO on the toxicological endpoint "immobilisation" give an EC50-24h= 372.3 mg/L [308.2 - 451.8] and an EC50-48h = 139.4 mg/L [116.3 - 166.01 based on nominal test item concentrations.

The test item METHYL BAPO is considered to be stable for all nominal concentrations because the measured concentrations of Total Carbon (TC) did not vary by more than 20% of the initial concentrations during the test.

The concentrations are expressed in nominal mg "METHYL BAPO loading rate" / L.

All the validity criteria were successfull.

Description of key information

The toxic effect of the registered substance to the invertebrate Daphnia magna was investigated in a preliminary and a definitive test under closed static conditions using Water Accommodated Fractions.

During the preliminary test, no effect was observed at the highest tested nominal concentration of 100 mg/L (loading rate).

During the definitive test, based on nominal loading rates, the EC50 based on immobility were determined to be of 372.3 mg.L-1 after 24h and of 139.4 mg.L-1 after 48h .

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

139.4 mg/L

Additional information

The aim of this study was to assess the effects of the test item METHYL BAPO on the daphnidsDaphnia magna, according to the OECD 202 guideline, in static conditions.

This study was carried out in accordance with the study planno.17/1228F/f dated September 12,2018.

There was no deviation and no amendment to the study plan during this study.

The concentrations which immobilise 50% of the daphnids exposed to the test item after 24 and 48 hours, reported as EC50 -24h and EC50-48h were evaluated, relative to the tested concentrations.

The results obtained for the test item METHYL BAPO on the toxicological endpoint "immobilisation" give an EC50-24h= 372.3 mg/L [308.2 - 451.8] and an EC50-48h = 139.4 mg/L [116.3 - 166.01 based on nominal test item concentrations.

Chemical analyses of the test item in the test solutions was performed by a Total Organic Carbon (TOC) analyzer in order to check the test item stability throughout the study.

The test item METHYL BAPO is considered to be stable for all nominal concentrations because the measured concentrations of Total Carbon (TC) did not vary by more than 20% of the initial concentrations during the test.

The concentrations are expressed in nominal mg "METHYL BAPO loading rate" / L.

All the validity criteria were successfull.