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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study is performed according to the OECD 201 guideline.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

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) and at the end of the test (t=72h).
- Concentrations: Samples for analysis will be taken from all test concentrations (WAFs) and control.
- Number of samples: Sampling will consist of single 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 loading rates. TOC analysis will not be performed in compliance with the 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 test item is not soluble in the algae test medium, a special solubilization protocol was carried 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, at obscurity and 20±2°C (these concentrations were prepared by adding directly the test item in flasks containing test medium). All the test solutions plus WAF Control (algae test medium) were then filtered through a membrane filter (cellulose acetate 0.45 μm) in order to remove suspended particles: solutions obtained are named WAF (Water Accommodated Fraction).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Species: Pseudokirchneriella subcapitata, CCAP 278/4
- Origin: Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Reason for selection: This system is a unicellular algal species susceptible to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
- Stock culture: Algae stock cultures are started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions are continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.
- Pre-culture: 2 to 4 days before the start of the test, cells from the algal stock culture are inoculated in test water at a cell density of 1.10^4 cells.mL-1. The pre-culture is maintained under the same conditions as used in the test. The cell density is measured immediately before use.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

No data

Test temperature:

23°C ± 1°C

pH:

Range-finding test: 7.9-8.3

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Range-finding test: 0, 1, 10 and 100 mg/L WAF

Details on test conditions:

TEST PROCEDURE AND CONDITIONS
- Test vessels: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace. Each test vessel will be uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Cell concentration: An initial cell density of 5.10^3 using an exponentially growing pre-culture.
- Test environment: Controlled environment cabinet (23°C ± 2°C); vessels will be distributed randomly in the incubator and redistributed over the test at t=24h and t=48h. During incubation, the algal cells will be kept in suspension by continuous magnetic stirring.
- Light regime/intensity: 6347 Lux; continuous illumination using cool white light with a light intensity of 60-120 μE.m-2.s-1 (equivalent range: 4440-8880 lux).
- Replicates: 3 controls and 2 replicates of each loading rate for counting. Moreover, replicates of each treatment were prepared for chemical analyses.

TEST WATER
- Standard medium used: yes: Original medium of OECD TG 201, Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1

EFFECT PARAMETERS MEASURED :
- Cell densities: Cell numbers will be counted daily by microscope using a counting chamber.
- pH: At the start and the end of the test in at least one vessel per treatment.
- Temperature of medium: Measured continuously in the growth chamber.
- Light intensity: Light intensity was measured once (t=0h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

DATA HANDLING
- Defining exposure concentrations: Effective concentrations were determined considering nominal WAF concentrations (nominal loading rate values).
- Determination of algal growth inhibition (if applicable): average growth rate and yield inhibition
- Statistical analysis: The software used to perform the statistical analysis will be ToxRat® Professional. Optionally, other statistical analyses may be performed if appropriate. EL10, EL20 and EL50 will be calculated (including 95% confidence interval) when possible for both growth rate and yield. When possible, LOELR and NOELR will be determined for both growth rate and yield.

RANGE-FINDING TEST
The range-finding test is carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 1, 10, and 100 mg.L-1 and to a control, according to two methods of preparation: use of solvent; and heating/sonication. The results of this preliminary test will be used to determine the range of loading rates to be used in the definitive study.

Reference substance (positive control):

not specified

Duration:

72 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:

growth rate

Remarks on result:

other: preliminary study

Details on results:

- No inhibition growth rate (0.1%) was observed at 100 mg test item /L during the preliminary test.
- Refer Tables 6.1.5/1, 6.1.5/2 and 6.1.5/3 for details

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

No data

 Table 6.1.5/1: Algae concentrations during the preliminary test

Nominal test item concentration		[algae] *10^4/mL at 24 hours			[algae] *10^4/mL at 48 hours			[algae] *10^4/mL at 72 hours
		Count.	Average	Cv%	Count.	Average	Cv%	Count.	Average	Cv%
Control (filtered)	a	3.6	4.0	11.5	24.9	22.3	10.5	147.9	148.9	6.2
	b	3.9			21.5			140.2
	c	4.5			20.4			158.6
WAF 1mg/L	a	4.3	4.8	14.7	24.4	24.5	0.6	154.6	170.5	13.2
	b	5.3			24.6			186.4
WAF 10mg/L	a	4.0	4.3	9.9	20.4	19.3	8.4	159.2	154.9	3.9
	b	4.6			18.1			150.6
WAF 100mg/L	a	3.7	4.0	10.6	20.3	21.8	9.4	134.1	148.4	13.6
	b	4.3			23.2			162.7

Cv: Coefficient of Variation

Table 6.1.5/2: Summary of growth rate inhibition and yield inhibition in the preliminary test

Nominal test item concentration		Algae *10^4/mL at 0 hour	Average growth rate μ0-72h	Inhibition growth rate (%)% Ir	Average inhibition growth rate (%)	Coefficient of variation	Yield 0-72h (algae *10^4/mL)(Y)	Average yield (algae*10^4/mL)	Yield inhibition (%) %Iy
									Per replicate	Average per replicate
Control (filtered)	a	1	1.666	Not applicable		1.2%	146.9	147.9	Not applicable
	b		1.648				139.2
	c		1.689				157.6
WAF 1mg/L	a	1	1.680	- 0.8	- 2.7	Not calculated	153.6	169.5	- 3.9	- 14.6
	b		1.743	- 4.5			185.4		- 25.4
WAF 10 mg/L	a	1	1.690	- 1.3	- 0.8	Not calculated	158.2	153.9	- 7.0	- 4.1
	b		1.672	- 0.2			149.6		- 1.1
WAF 100mg/L	a	1	1.633	2.1	0.1	Not calculated	133.1	147.4	10.0	0.3
	b		1.697	- 1.8			161.7		- 9.3

 

Table 6.1.5/3: Daily average growth rate of control

Nominal test item concentration		Specific growth rate 0-24h μ0-24h	Specific growth rate 24 -48h μ24-48h	Specific growth rate 48 –72h μ48-72h	Mean of Specific growth rate 0-72h	Cv (%)	Mean of Cv
Control (filtered)	a	1.281	1.934	1.782	1.666	20.5	18.3%
	b	1.361	1.707	1.875	1.648	15.9
	c	1.504	1.511	2.051	1.689	18.6

 

Table 6.1.5/4: Validation of test

	Values for OECD 201	Validity of the preliminary test
Increase of the control biomass	> 16	Yes (148.9)
Average specific growth rate CV of controls	< 7%	Yes (1.2%)
Daily average growth rate CV of controls	< 35%	Yes (18.3%)

 

Validity criteria fulfilled:

yes

Remarks:

refer Table 6.1.5/4

Conclusions:

Under the test conditions, no inhibition growth rate (0.1%) of algae was observed at 100 mg/L WAF of the test item during this preliminary test. Therefore, the EL50 of the test item is considered as >100 mg/L WAF.

Executive summary:

A preliminary study was performed to assess the test item for its ability to generate toxic effects on the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Algal cells were exposed to 0, 1, 10 and 100 mg/L Water Accommodated Fractions (WAFs) of the test item. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material in the control and the test loading rates was checked by TOC analysis at the start and at the end of the test.

Under the test conditions, no inhibition growth rate (0.1%) of algae was observed at 100 mg/L WAF of the test item during this preliminary test. Therefore, the EL50 of the test item is considered as >100 mg/L WAF.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-09-18 to 2019-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not relebvant.

GLP compliance:

yes (incl. QA statement)

Remarks:

Signeg 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 algal test solutions was performed according to the Analytical protocol FCBA Chimie No.410-e version 2: Method for the chemical analysis of Total Carbon (TC) in samples contained in the test item METHYL BAPO from algae ecotoxicology solutions by a Total Organic Carbon Analyzer and the following protocol:
- At the beginning of the test (T = 0h)
- At the end of the test (T = 72h)

Schedule analysis at initial time (T0h):
-T0h Control
-T0h 10 mg/L
-T0h 102 mg/L
-T0h 1050 mg/L
All the solutions were analysed taken directly from the starting solutions without algae.
Schedule analysis at final time (T72h):
-T72h Control Abiotic
-T72h 10 mg/L Abiotic
-T72h 102 mg/L Abiotic
-T72h 1050 mg/L Abiotic

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:

WAF preparation: 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.
According to the study monitor and the results of the preliminary test, this study was conducted on five concentrations levels: 10 - 32 - 102 - 328 and 1050 mg/L.
The test solutions plus Control (algal test medium) were then filtered to remove suspended particles: solutions obtained are named WAF (Water Accommodated Fraction).

Test organisms (species):

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

Details on test organisms:

Strain number CCAP 278.4
The algae cultures are storêd in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular
sub-culturing (every week).

A growing exponential phase inoculum of the freshwater unicellular alga Pseudokirchneriella subcapitata was added in order to have an initial concentration of (10)^4 cells/ml at the start of the test: - 100 mL of algal cultures grown in 250 mL erlenmeyer flasks which were placed in a cooling incubator at a temperature in the range of 21 to 24°C controlled at t 2°C and under a constant shaking fixed at 120 rpm and a continuous illumination comprised between 4 440 and I 880 lux.

Algae measurement: Beckman Coulter 22 and Malassez counting cell

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

22.7 °C - 23.2°C

pH:

7.9

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal concentration: 10, 32, 102, 328, 1050 mg/L (loading rates)

Details on test conditions:

TEST SYSTEM
Control: 6 biotic replicates + 1 abiotic replicate .
Concentration 10 mg/L: 3 biotic replicates + 1 abiotic replicate .
Concentration 32 mg/L: 3 biotic replicates .
Concentration 102 mg/l:3 biotic replicates + 1 abiotic replicate .
Concentration 328 mg/L: 3 biotic replicates .
Concentration 1050 mg/L: 3 biotic replicates + 1 abiotic replicate

GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Light intensity and quality: 6584 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beckman Coulter 22 and Malassez counting cell

TEST CONCENTRATIONS
- 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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 050 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

432 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate

Remarks:

[347 - 518 mg/L]

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

588 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yied

Remarks on result:

other: loading rate

Remarks:

[525 - 663 mg/L]

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: loading rate

Details on results:

ln the control, the increase of the biomass during 72 hours was determined to be higher than factor of 16.
The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

Results with reference substance (positive control):

The last algalgrowth inhibition test performed with the reference item KzCrzOz (June 11,2018) gave an ErCso(O-72h) =1.12 mg/L (1.04 - 1.20), indicating that the algal sensitivity is correct (within the accepted interval of 0.65 to 1.73 mg/L fixed by the lntemational Standard NF EN ISO 8692 from Mai 2012 as mentioned in the OECD Guideline) and in accordance with previous data obtained by the laboratory.

Reported statistics and error estimates:

One deviation was observed in this Study. Additonal abiotic flasks were added to the test design : Control, 10,102 and 1050 mg/L for the chemical analyses at T0h and T72h in order to evaluate the stability of the test item without algae due to the analysis of Total carbon.
This deviation has no incidence on study results.

pH values and observations

Nominal test item loading rate (mg/L )	pH at the start (biotic replicates)	pH after 72 hours (biotic replicates)
Control	7.9	8.1
10	7.8	7.9
32	7.8	8.8
102	7.8	8.6
328	7.9	7.8
1050	7.9	7.7

Raw data, i.e. algal concentrations values expressed as cell number *l0⁴ per mL of test solutions after 24, 48 and 72 hours of incubation are indicated in the following table.

Nominat test item loading rate		[algae] *10^4/mL at 24 hours			[algae] *10^4/mL at 48 hours			[algae] *10^4/mL at 72 hours
		Count.	Average	cv	Count.	Average	cv	Count.	Average	cv
Control	a	4,8	5	9,8	26,0	30,7	12,6	224,4	244,4	4,6
	b	4,4			26,3			247,5
	c	5,4			33,7			252,7
	d	4,8			30,0			256,2
	e	4,7			32,9			242,3
	t	5,7			35,0			243,2
10 mg/L	a	4,8	4,6	9,5	33,4	30,4	10,8	258,1	238.7	8.2
	b	4,9			30,9			219,0
	c	4,1			26,9			239,0
32 mg/L	a	5,3	4,8	9,1	29,6	26,7	9,4	228,8	206.8	10,1
	b	4,5			25,3			204,5
	c	4,6			25,2			187,2
102 mg/L	a	4,7	4.8	10,6	27,6	29,5	5,7	224,6	207.9	7.1
	b	4,3			30,9			202,8
	c	5,3			29,9			196,3
328 mg/L	a	3,7	3,8	9,5	29,5	27,5	10,8	154,0	163	10.6
	b	3,5			24,1			152,1
	c	4,2			28,9			182,9
1050 mg/L	a	4,1	3,6	12,4	14,9	13,4	12,0	47,2	44	6.2
	b	3,6			13,5			42,3
	c	3,2			11,7			42,6

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item "METHYL BAPO" on the toxicological endpoint "inhibition of growth rate" indicate an ErC50-(0-72h) higher than 1050 mg/L (highest tested concentration) value expressed in nominal test item concentration.

Executive summary:

The aim of this study was to determine the effects of the test item "METHYL BAPO" on the growth of the unicellular green alga Pseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

The results obtained for the test item "METHYL BAPO" on the toxicological endpoint "inhibition of growth rate" indicate an ErC50-(0-72h) higher than 1050 mg/L (highest tested concentration) value expressed in nominal test item concentration.

The results obtained for the test item "METHYL BAPO" on the toxicological endpoint "Yield inhibition" indicate an EyC50 -(0-72h)= 588 mg/L [525 - 663], value expressed in nominal test item concentration.

ln the control, the increase of the biomass during 72 hours was determined to be higher than factor of 16.

The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

The validity criteria were successfully fulfilled.

Description of key information

The toxic effect of the registered substance to the unicellular algal species Pseudokirchneriella subcapitata 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 72-hour EL50 for the parameters growth rate and yield were determined to be >1050 mg.L-1 and 588 mg.L-1, respectively. The 72-hour EL10 for growth rate was 432 mg L-1 and for yield was 29 mg.L-1.

Key value for chemical safety assessment

EC50 for freshwater algae:

1 050 mg/L

EC10 or NOEC for freshwater algae:

432 mg/L

Additional information

During the definitive study the effects of the test item "METHYL BAPO" on the growth of the unicellular green alga Pseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011) were determined.

The results obtained for the test item "METHYL BAPO" on the toxicological endpoint "inhibition of growth rate" indicate an ErC50-(0-72h) higher than 1050 mg/L (highest tested concentration) value expressed in nominal test item concentration.

The results obtained for the test item "METHYL BAPO" on the toxicological endpoint "Yield inhibition" indicate an EyC50 -(0-72h)= 588 mg/L [525 - 663], value expressed in nominal test item concentration.

ln the control, the increase of the biomass during 72 hours was determined to be higher than factor of 16.

The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

The validity criteria were successfully fulfilled.