Dialkyl C18 and C18-unsaturated phosphonates

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-06-09 to 2017-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2015-11-03

Test material

Test material information

Constituent 1

Reference substance name:

Dialkyl C18 and C18-unsaturated phosphonates

EC Number:

701-298-1

Cas Number:

64051-29-2

Molecular formula:

Not applicable for a UVCB Substance

IUPAC Name:

Dialkyl C18 and C18-unsaturated phosphonates

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: 1 mL were taken in all test groups at the following time: 0h, 24h and 72h.
- Sample storage conditions before analysis: in a freezer (≤-15°C) until analysis

Test solutions

Vehicle:

yes

Remarks:

Acetone (Only in the final test)

Details on test solutions:

The batch of Alkenyl phosphonate tested was a colourless liquid and a UVCB substance. No correction was made for the purity/composition of the test item.

For the combined limit/range-finding test, preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A 3- day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of 1 hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.

For the final test, preparation of test solutions started with a stock solution of 100 mg/L in acetone. A 15- minute period of magnetic stirring was applied to ensure maximum dissolution of the test item in acetone. Thereafter, 100 µL of the stock was spiked into 1 litre of medium to reach a test item concentration of 10 µg/L.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: strain: NIVA CHL 1
- Source: In house laboratory culture
Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3 /L

Test temperature:

22°C - 23°C

pH:

T0: 8.5 to 8.6
T72h: 7.7 to 7.8

Dissolved oxygen:

No data

Salinity:

Not applicable: Fresh water

Nominal and measured concentrations:

final test: 10µg/L which coresponfd to the concentration exceeding the water solubility of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100mL all glass
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: the vessels contain 50 mL of test solution
- Aeration: No
- Initial cells density: 10 e 4 cell / mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 81 to 84 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED 4.9.1.

Recording of Cell Densities
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.


TEST CONCENTRATIONS
- Range finding study: 1, 10 and 100 mg/L
- Test concentrations: 10 µg/L

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.67 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.67 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.67 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Any other information on results incl. tables

Mean Cell Densities (x10⁴Cells/mL) During the Combined Limit/Range-Finding Test

Time (h)	Alkenyl phosphonate; Loading rate (mg/L)
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	7.2	n.d.	n.d.	2.8
48	56.3	n.d.	n.d.	3.5
72	297.6	296.3	272.8	4.4

n.d. not determined

Percentage Inhibition of Growth Rate During the Combined Limit/Range-Finding Test

Alkenyl phosphonate Loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.898	0.0195	6
1.0	1.897	0.0221	3	0.1
10	1.869	0.0314	3	1.6
100	0.480	0.0965	6	74.7

Growth Rate And Percentage Inhibition For The Total Test Period (Main test)

Alkenyl phosphonate TWA concentration (µg/L)	Mean	Std. Dev.	n	%Inhibition
Solvent-control	1.822	0.0173	6
0.67	1.825	0.0208	6	-0.2

 

Growth Rate And Percentage Inhibition At Different Time Intervals

Alkenyl phosphonate TWA concentration (µg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Solvent-control	6	1.858		1.970		1.638
0.67	6	1.784	3.9	2.045	-3.8	1.646	-0.5

 

Effect Parameters

Parameter (µg/L)	NOEC	EC10	EC20	EC50
Growth rate	0.67	>0.67*	>0.67*	>0.67*
Yield	0.67	>0.67*	>0.67*	>0.67*

* 95% confidence intervals could not be determined

pH Levels Recorded During the Limit Test

Alkenyl phosphonate TWA concentration (µg/L)	pH
	t=0h	t=72h
Blank-control	8.5	7.8
Solvent-control	8.6	7.8
0.67	8.6	7.7

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of Alkenyl phosphonate tested.
The EC50 for growth rate inhibition (72h-ERC50) and yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded a TWA concentration of 0.67 µg/L.
The 72h-NOEC for both growth rate inhibition and yield inhibition was 0.67 mg/L.
Due to the very low solubility of Alkenyl phosphonate in water, concentration levels that might be toxic for algae could not be reached.
It should be noted that the concentration of 0.67 µg/L is considered to exceed both the water and medium solubility of the test item.

Executive summary:

The objective of the study was to evaluate Alkenyl phosphonate for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10 and EC50 for both inhibition of growth rate and inhibition of yield.

 

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

 

The batch of Alkenyl phosphonate tested was a colourless liquid and a UVCB substance.

Stock solution was prepared in acetone at a factor 10,000 higher than the final test concentration. Thereafter, 100 µL of the stock was spiked into 1 litre of medium to reach a test item concentration of 10 µg/L.

 

A final test was performed as a limit test, based on the results of a preceding combined limit/range-finding test and the information about solubility of test item in water.

 

Six exponentially growing algal cultures were exposed to an untreated control, a solvent control and to the nominal concentration of 10 µg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

 

At the start of the test, the actual test concentration was 13 µg/L (133 % of nominal). After 24hours, the measured concentration had decreased below the limit of detection of the analytical method, i.e. below 0.39 µg/L. The calculated Time Weight Average (TWA) concentration was 0.67 µg/L.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

 

Parameter (µg/L)	NOEC	EC10	EC20	EC50
Growth rate	0.67	>0.67*	>0.67*	>0.67*
Yield	0.67	>0.67*	>0.67*	>0.67*

* 95% confidence intervals could not be calculated

 

Due to the very low solubility of Alkenyl phosphonate in water, concentration levels that might be toxic for algae could not be reached.

 

In conclusion, the EC50 for growth rate inhibition (72h-ERC50) and yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded a TWA concentration of 0.67 µg/L.

 

The 72h-NOEC for both growth rate inhibition and yield inhibition was 0.67 µg/L. It should be noted that the concentration of 0.67 µg/L is considered to exceed both the water and medium solubility of the test item.