Bis(2-ethylhexyl) phosphonate

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:

12 Dec 2017 - 2 Feb 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Physical effects cannot be excluded since daphnids were observed to be at the surface of the test solutions at concentrations relevant for ECx calculation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) phosphonate

EC Number:

222-904-6

EC Name:

Bis(2-ethylhexyl) phosphonate

Cas Number:

3658-48-8

Molecular formula:

C16H35O3P

IUPAC Name:

bis(2-ethylhexyl) phosphonate

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Analytical samples taken at 0 hours (initial value) and 24 hours from fresh and 24 hours and 48 hours from aged test solutions were analysed from control and all test item concentrations. For each sampling also retain samples were taken.
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solutions for the three highest nomical concentrations were prepared by directly weighing 10.0, 4.55, and 2.07 mg in 1000 mL test medium respectively. These stock solutions were stirred at room temperature for about 24 h in the dark and in a closed vessel and let settle down for 1 h (based on OECD Series on Testing and Assessment No. 23). After the settling, the necessary volumes for the test were withdrawn via a Teflon tube from the medium level of the stock solution. The two lowest nominal concentrations were prepared by diluting the appropriate solutions to give the required nominal concentrations. The preparation procedure was repeated after 24 hours. Approx. 50 mL of the prepared solutions were transferred to each test vessel.
- Controls: blank control without the test item
- Eluate: no
- Differential loading: yes, except for the twp lowest test concentrations
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Small oil droplets were observed at the water surface in the stock solutions with the two highest concentrations

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: Continuously bred in the laboratory and originally purchased in a healthy condition from the Federal Environment Agency in Berlin/Germany.
- Age: Freshly hatched daphnids less than 24 hours old were used for the test.
- Culture conditions: Daphnia magna was bred as single culture (1 daphnid per 100 mL) in Elendt M4 medium. The pH value of the aerated water was within a range of 6.0 – 9.0. The dissolved oxygen was above 60 % saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH. The animals were fed with single cell green algae (Desmodesmus subspicatus, formerly Scenedesmus subspicatus) at least three times a week.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

12°dH (214 mg/L as CaCO3)

Test temperature:

19.5 ± 0.3 °C

pH:

7.94 ± 0.08

Dissolved oxygen:

8.8 ± 0.1

Nominal and measured concentrations:

nominal: 10.0, 4.55, 2.07, 0.939, 0.427 mg/L and control
measured: 0.500, 0.364, 0.145, 0.0657, 0.0299 mg/L and < LOD

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel (100 mL), were filled up with ~ 50 mL test solution. The test units were covered with a glass plate.
- Renewal rate of test solution: The test solutions were freshly prepared at 0 and 24 h.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Elendt M4 standard medium was used as test medium. At test initiation the pH-value of the control (untreated test medium) was 7.81, the dissolved oxygen concentration was 8.9 mg/L and the total hardness was 12°dH (214 mg/L as CaCO3).
- Intervals of water quality measurement: The test temperature and the pH-value as well as the oxygen concentration of the test solutions were measured at all concentrations and the control at t = 0 h fresh and t = 24 h from fresh test solutions and after t = 24 h and t = 48 h from aged test solutions in one separate replicate per concentrations without test organisms.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod/8 hours darkness daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobility after 24 and 48 h
- Behavioural changes of daphnids were recorded at 24 and 48 hours after starting the test.

RANGE-FINDING STUDY
- A range-finding study was conducted but the results are not reported.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.121 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 0.025 - 0.549 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.066 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: 0%
- Behavioural changes: After 24 and 48 hours some of the mobile daphnids at the nominal test concentrations 0.427 and 0.939 mg/L were found at the surface of the test solution. At the test concentrations 2.07, 4.55, and 10.0 mg/L, all daphnids were found at the surface.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Small oil droplets were observed at the water surface in the stock solutions with the two highest test concentrations.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 (24 h) < 1.00 mg/L

Reported statistics and error estimates:

The 24 h and 48 h EC50 are the calculated concentrations where 50 % of the daphnids were immobilised after 24 and 48 hours, respectively. The values for EC50 were determined by Probit analysis using max. linear likelihood regression. The evaluation of data was performed by ToxRat Professional 3.2.1. The NOEC was established based on the highest concentration at which the immobilisation is not higher than the allowed control immobilisation (≤ 10 % immobilisation).

Any other information on results incl. tables

Table 1: Biological results of the test, 48 h values

	Nominal concentration [mg/L]
	Control	0.427	0.939	2.07	4.55	10.0
	Immobilised daphnids after 48 h
Group 1	0	0	1	1	5	5
Group 2	0	1	0	3	5	5
Group 3	0	0	0	4	5	5
Group 4	0	0	0	4	5	5
Σ	0	1	1	12	20	20
%	0	5	5	60	100	100

Table 2: Determined concentration of the test substance

Test item	Sampling	Test item		Geometric mean	Actual concentration
nominal [mg/L]	[h]	[mg/L]	% of nominal	[%]	[mg test item/L]
Control	0 fresh	n.d.	-	-	-
	24 aged	n.d.	-
	24 fresh	n.d.	-
	48 aged	n.d.	-
0.427	0 fresh	0.0569	13	7	0.0299
	24 aged	0.0224	5
	24 fresh	0.0455	11
	48 aged	0.0166	4
0.939	0 fresh	0.120	13	7	0.0657
	24 aged	0.0413	4
	24 fresh	0.0907	10
	48 aged	0.0350	4
2.07	0 fresh	0.275	13	7	0.145
	24 aged	0.099	5
	24 fresh	0.212	10
	48 aged	0.0873	4
4.55	0 fresh	0.594	13	8	0.364
	24 aged	0.232	5
	24 fresh	0.577	13
	48 aged	0.235	5
10.0	0 fresh	0.845	8	5	0.500
	24 aged	0.394	4
	24 fresh	0.745	7
	48 aged	0.320	3

 - = not calculated; n.d. = not detectable; LOQ = 0.0100 mg/L test item

Table 3: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.	Control: 0%	yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	≥ 8.7 mg/L	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.