Dialkyl C18 and C18-unsaturated phosphonates

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-06-09 to 2017-10-05

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 202 (Daphnia sp. Acute Immobilisation 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:

Single samples for possible analysis were taken from all test groups according to the schedule below.
Frequency at t=0 h and t=48 h
Volume 1.0 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 1.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for possible analysis until delivery of the final report with a maximum of three months

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
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 of 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, 1820
- Source: In-house laboratory culture with a known history.
- Age of parental stock (mean and range, SD): < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: No

ACCLIMATION
- Acclimation period: In house laboratory culture
- Type and amount of food: a suspension of fresh water algae.
- Feeding frequency: Daily

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

20 °C to 21 °C

pH:

8.0 to 8.3

Dissolved oxygen:

8.8 mg/L to 9 mg/L

Salinity:

Not applicable, fresh water

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass vessel
- Material, size, headspace, fill volume: 60 mL vessels containing 50 mL of test solution
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Medium M7, as prescribed by Dr. Elendt-Schneider
(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts: CaCl2.2H2O 2 11.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements:
B 0.125 mg/L
Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rb and Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025 mg/L
Se 0.0010 mg/L
V 0.0003 mg/L
Na2EDTA.2H2O 2.5 mg/L
Macro nutrients:
Na2SiO3.9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L
The hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h hour light per day

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


RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/L (nominal concnetration)
- Results used to determine the conditions for the definitive study:No significant immobility (i.e. >10%) was observed in the control and the two lowest concentrations tested during the test period. At the highest concentration, 75% of daphnids were immobilised at the end of the exposure. It should be noted that all the daphnids in the control treatment were trapped at the surface of test solution. The results of this test were accepted as in two parallel performed tests with daphnids from the same batch, no such effects were observed.
Samples taken from WAFs prepared at a loading rate of 10 and 100 mg/L were analysed. The actual concentrations were 0.15 and 2.2 mg/L at the start of the test, respectively. Measured concentrations decreased to 2.9% and 10% of initial at the end of the test, respectively.
The measured concentrations strongly exceeded solubility of the test item in water even in groups were no immobility was observed (i.e. WAF of 10 mg/L). Therefore, it was decided to perform the final test as a limit with concentration exceeding water solubility of the test item by factor not exceeding 100, i.e. 10 µg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 4.5 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: No
- Other adverse effects control: No
- Abnormal responses: No
- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Dose-response test: Yes
- 48h-EC50: 0.52-0.66 mg/L

Any other information on results incl. tables

Number of Introduced Daphnids and Incidence of Immobility in the Combined Limit/Range-Finding Test

Time (h)	Replicate	Alkenyl phosphonate; loading rate (mg/L)
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0(5)	0	0	0
	B	0(5)	0	0	0
	C	0(5)			0
	D	0(5)			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0
48	A	1(5)	0	0	2
	B	0(5)	0	0	4#
	C	0(5)			3
	D	0(5)			4
	Total immobilised	1	0	0	13
	Effect %	5	0	0	75

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility,^#Microscopic observation revealed no test item attached to the daphnids,

 

Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)	Replicate	Alkenyl phosphonate; Average exposure concentration (µg/L)
		Blank-control	Solvent-control	4.5
0	A	5	5	5
	B	5	5	5
	C	5	5	5
	D	5	5	5
	Total introduced	20	20	20
24	A	0	0	0
	B	0	0	0
	C	0	0	0
	D	0	0	0
	Total immobilised	0	0	0
	Effect %	0	0	0
48	A	0	0	0
	B	0	0	0
	C	0	0	0
	D	0	0	0
	Total immobilised	0	0	0
	Effect %	0	0	0

 

 

 

Effect Parameters

Parameter	Alkenyl phosphonate Average exposure concentration (µg/L)
24h-EC50	>4.5*
48h-EC50	>4.5*

* - 95% confidence intervals couldnot be determined

 

pH and Oxygen Concentrations (mg/L) During the Final Test

Alkenyl phosphonate Average exposure concentration (µg/L)	Start (t=0 h)		End (t=48 h)
	pH	O2	pH	O2
Blank-control	8.0	8.9	8.3	9.0
Solvent-control	8.0	8.8	8.3	9.0
10	8.0	8.9	8.3	9.0

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 for Daphnia magna exposed to Alkenyl phosphonate was beyond the range tested, i.e. exceeded the average exposure concentration of 4.5 µg/L (this concentration was consider to exceed solubility of the test item in both medium and water).

Executive summary:

The objectiveof the study was to evaluate Alkenyl phosphonate for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC₅₀at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004.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 information about water solubility. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control, a solvent control and to 10 µg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Analysis of the samples taken at the start of the final test showed a measured concentration of 9.84 µg/L (98% of the nominal concentration). During the exposure period the measured concentrationhad decreased to 2.04µg/L (21 % of initial). The calculated average exposure concentration was 4.5 µg/L.

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

In conclusion, the 48h-EC₅₀for Daphnia magna exposed to Alkenyl phosphonate was beyond the range tested, i.e. exceeded the average exposure concentration of 4.5µg/L (this concentration was consider to exceed solubility of the test item in both medium and water).