N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane-2-oxide-2-yl)ethane-1,2-diamine

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:

June 4, 2013 to June 7, 2013

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)

Version / remarks:

OECD Guidelines for Testing of Chemicals, No.201, March 23, 2006, Annex 5 corrected: July 28, 2011, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Japanese National Test Guidelines

Version / remarks:

"Algal growth inhibition test" stipulated in the "Test Methods of the New Chemical Substances etc." (March 31, 2011, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, partly amended by Yakushokuhatsu 0402 No.1, Heisei 24.03.28 Seikyoku No.2, Kanpokihatsu No. 120402001 on April 2, 2012)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

The sample taken from the test solution was diluted to make the final composition of methanol/medium (1/1 v/v) to prepare samples for high performance liquid chromatography (HPLC).

Vehicle:

no

Details on test solutions:

Required amount of the provided substance and the medium were mixed to make a 100 mg/L solution. Then the test solution was prepared by stirring for 48 hours, and then distributed into test vessels.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata
Reason for selection of the species: Use of the species is recommended in the test guidelines.
Source of strain: American Type Culture collection
Strain number: ATCC 22662
Acquisition date: June 30, 1995
Keeping organisms after acquisition: Organisms are subcultured in a sterile condition in Kurume Laboratory.

Confirmation of reproducibility of the test system: Algal growth inhibition tests are periodically performed with the reference substance. The latest data is shown below.
Reference substance: potassium dichromate (special grade, Lot No. HLH7646, Wako Pure Chemical Industries, Ltd.)
Test period: From December 10, 2012 to December 13, 2012
ErC50 (days 0-3): 1.2 mg/L
This value was within the determined range of the background data in Kurume Laboratory, which is a range of “mean ± 2 × standard deviation” [mean ± standard deviation: 0.93 ± 0.17 mg/L (n = 21)].

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified

Hardness:

Not specified

Test temperature:

21 -24 °C (controlled at ±2 °C)

pH:

7.9

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

100 mg/L nominal

Details on test conditions:

Test equipment and apparatus
Test vessel: 500 mL glass Erlenmeyer flask (with an air-permeable stopper, SILICOSEN®)
Culturing apparatus: An apparatus enabling to conduct the culture by continuous shaking and the control of temperature and lighting (constant temperature shaking incubator with illumination, USI Corp.)

Test conditions
Exposure system: Culturing by gyratory shaking (approximately 100 rpm)
Exposure period: 72 hours
Test concentration: 100 mg/L (as a limit test at the upper concentration limit for used test method)
This test concentration was determined based on the result of the preliminary study.
The result of the preliminary study is shown in Additional data.
Control group: Medium without the test substance which was treated by the same preparation method for the test solution.
Number of the replicates: 6 replicates/test group
Volume of the test solution: 600 mL/test group (100 mL/test vessel)
The number of cells at the start of exposure: The number of cells which was pre-cultured for three days from June 1, 2013 to June 4, 2013 was counted, and then inoculated into the test solution to make 0.75 × 104 cells/ml of the cell concentration in the test solution.
Test operation: The test was conducted by aseptic manipulation.
Temperature: 21 -24 °C (controlled at ±2 °C)
Lighting: Nominal value 90 μE/m2/s (within ± 20% of the nominal value, and controlled variance at the mean value ± 15 °C), continuous lighting by fluorescent lamps with a spectrum range of 400 to 700 nm.)

Observation and measurement
Algal growth, etc.
Measurement items: Biomass (cell concentration)
Frequency of measurement: Every 24 hours after the start of exposure (blank correction was performed by measuring a background value for the test solution.)
Observation of cells: One test vessel per each test group was observed at the end of exposure.
Measurement instrument: Coulter Counter Z1, Beckman Coulter, Inc. Biological microscope, BX41, Olympus Co., Ltd.

Condition of the test solution
Condition of the test solution was observed at the start and end of exposure.

Quality of the test solution and exposure environment
pH: The pH of an aliquot from the preparation vessel was measured (at the start of exposure).
The pH values of all test vessels of the test groups were measured because two or more times difference of the biomass and two or more times difference of the growth inhibition rate were observed in the test vessels of the test groups at the end of exposure.
Temperature in incubator: The temperature was measured once per day during exposure period.
Light intensity: The light intensity in the incubator was measured once per day during exposure period.
Instruments: Portable pH meter, HM-21P, TOA D.K.K Certified glass stem thermometer Light quantum meter, LI-250A, LI-COR Inc.

Test substance concentration in the test solution
Frequency of measurement: At the start and end of exposure.
Sampling methods: Separately sampled from the preparation vessel at the start of exposure. An aliquot was sampled from the test vessels of each test group at the end of exposure. Further, the concentrations of the test substance of all test vessels of the test groups were measured because two or more times difference of the biomasses and two or more times difference of the growth inhibition rate were observed in the test vessels of the test groups at the end of exposure.
Removal of algae: Centrifugal separation (3000 rpm for 10 minutes) was conducted only at the end of exposure.
Sample amount: Approximately 50 - 90 mL for the control group, approximately 10 - 90 mL for the test group

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Observation of the test solution and measurement results
Conditions of the test solution
The test solutions of the control group and the test concentration group were colorless and transparent at the start of exposure. At the end of exposure, it was green due to cell growth (color of the solution in one vessel of the test concentration group was light green).

Water quality of the test solution and exposure environment
The pH of the test substance was 7.9. The temperatures in the incubator were 21.1 - 21.6 °C and the light intensities were 91 - 96 μE/m2/s.

Test substance concentrations in the test solution
The measured concentrations of the test substance in the test solution were 100 mg/L at the start of exposure and 100 mg/L at the end of exposure (vessel A: 100 mg/L, vessel B: 100 mg/L, vessel C: 99 mg/L, vessel D: 100 mg/L, vessel E: 98 mg/L and vessel F: 100 mg/L).
Further, these concentrations were 101% and 98 - 100% of the nominal concentration, and maintained within ± 20 % of the nominal concentration.

ErC50
ErC50 of the test substance calculated from the growth rates was > 100 mg/L.

Growth curve, cell observation result and NOEC for each test group
Algal growth in the test concentration group showed the similar level of algal growth as in the control group. As a result of cell observation, no abnormality was observed in the control group, and also no abnormality was observed in the test concentration group. As a result of the significance test for growth rates, no statistically significant difference was noted. Based on the results of the significance test and of the above-mentioned observation of the cells, NOEC calculated from growth rates was > 100mg/L.

Discussion
This test was carried out as the limit test to confirm the presence or absence of the effect of the test substance on test organisms at the upper concentration limit (100 mg/L) for used test method.
As a result, algal growth in one test vessel (vessel D) of the 100 mg/L group was remarkably low, and its inhibition rate was 25%. Algal growth in other five vessels showed a similar algal growth to the control group. In the measurement of the pH values and concentrations of the test substance in the test solution for every test vessels at the end of exposure, the measured values of the vessel D showed the similar values to other vessels. Therefore, it was considered that the effect shown in the vessel D was not caused by the test substance. According to these results, any effect on the test organisms was not observed, and it was considered that the test substance did not affect the test organisms at the upper concentration limit (100 mg/L) for used test method.
The test substance concentration in the test solution was maintained ± 20% of the nominal concentration, and the environmental conditions were within the proper range for the test. Therefore, it was considered that this study was in accordance with this test method.

Results with reference substance (positive control):

Not required

Reported statistics and error estimates:

See "Any other information" for details

pH of test solutions

Nominal concentration (mg/L)	pH
	At the start	At the end
Control	7.9	7.9
100	7.9	No.A:7.9, No.B:7.9; No.C:7.9, No.D:7.9, No.E:7.9, No.F:7.9

 

Culture temperature and light intensity in incubator

Time	At the start	1-day	2-day	At the end
Culture temperature (°C)	21.6	21.1	21.5	21.3
Light intensity (µEm^2/s)	96	92	91	93

 

Value of Biomass at each time

Nominal concentration (mg/L)	No.	Cell concentration (x10^4cells/mL)
		0 hour^a	24 hours	48 hours	72 hours
Control	A B C D E F	0.75 0.75 0.75 0.75 0.75 0.75	3.3 4.1 4.2 4.2 3.8 3.6	14 18 20 16 20 15	54^b 81 87 65 80 59
	Mean S.D.	0.75 0	3.9 0.35	17 2.6	71 14
100	A B C D E F	0.75 0.75 0.75 0.75 0.75 0.75	4.1 4.3 3.9 3.0 3.5 4.3	21 17 20 8.8 16 19	96 59 85 22 68 86
	Mean S.D.	0.75 0	3.9 0.52	17 4.5	69 26

^aThe value based on the measured value of pre-culture

^bThe minimum cell growth in control (biomass at the end of exposure / biomass at the start of exposure) 54 / 0.75 = 72

 

Growth rate and growth inhibition

Nominal concentration (mg/L)	No.	Growth rate (0-3d)	Inhibition rate (%)
Control	A B C D E F	1.42 1.56 1.59 1.49 1.55 1.45	- - - - - -
	Mean S.D.	1.51 0.0651	- -
100	A B C D E F	1.62 1.46 1.58 1.13 1.50 1.58	-6.9 3.6 -4.2 25 0.66 -4.5
	Mean S.D.	1.48 0.178	2.3 12

 

ErC50 and NOEC

ErC50 (mg/L)	NOEC (mg/L)
>100	≥ 100

 

Result of statistical analysis

Nominal concentration (mg/L)	Statistical analysis	Statistical procedure
100	n.s.	F test Aspin-Welch t-test

n.s.: no significant difference

 

Variation of growth rate in control

Variation for section-by-section specific growth rates in the controls

Control No.	Mean	Standard deviation	Coefficient of variation (%)
A	1.42	0.0749	5.3	8.6 (Mean)
B	1.56	0.121	7.8
C	1.59	0.130	8.2
D	1.49	0.198	13
E	1.55	0.157	10
F	1.45	0.103	7.1

 

Variation of average specific growth rates in replicate controls

	0-3 day
Mean	1.51
Standard deviation	0.0651
Coefficient of variation (%)	4.3

 

Validity criteria fulfilled:

yes

Conclusions:

EC50 (ErC50) > 100 mg/L
NOEC (growth rate, days 0-3) > 100 mg/L
(These concentrations are based on the nominal value.)

Executive summary:

Test organism: Pseudokirchneriella subcapitata

Test groups: 100 mg/L group and a control group

Preparation of test solutions: Prepared by mixing the test substance and the medium and then stirring for 48 hours.

Exposure system: Culturing by gyratory shaking (approximately 100 rpm)

Exposure period: 72 hours

Number of replicates: 6 replicates per test group

Volume of the test solution: 600 mL per test group (100 mL per test vessel)

Incubation temperature: 21.1 – 21.6 °C

Light intensity: 91 – 96 μE/m2/s

Measurement of biomass: Cell concentration

Analysis of the test substance in test solutions: HPLC method (at the start and end of exposure)

 

Test result

EC50 (ErC50) > 100 mg/L

NOEC (growth rate, days 0-3) > 100 mg/L

(These concentrations are based on the nominal value.)

Description of key information

EC50 (ErC50) > 100 mg/L

NOEC (growth rate, days 0-3) > 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Test organism: Pseudokirchneriella subcapitata

Test groups: 100 mg/L group and a control group

Preparation of test solutions: Prepared by mixing the test substance and the medium and then stirring for 48 hours.

Exposure system: Culturing by gyratory shaking (approximately 100 rpm)

Exposure period: 72 hours

Number of replicates: 6 replicates per test group

Volume of the test solution: 600 mL per test group (100 mL per test vessel)

Incubation temperature: 21.1 – 21.6 °C

Light intensity: 91 – 96 μE/m2/s

Measurement of biomass: Cell concentration

Analysis of the test substance in test solutions: HPLC method (at the start and end of exposure)

 

Test result

EC50 (ErC50) > 100 mg/L

NOEC (growth rate, days 0-3) > 100 mg/L

(These concentrations are based on the nominal value.)