Bis(2-ethylhexyl) phosphonate

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:

8 Jan - 8 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006, corrected 2011

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 were taken in the main test from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours and 72 hours from aged test solutions. Samples taken after 0 hours, 24 hours and 72 hours from the control and all test concentrations including and above 0.954 mg/L were analysed. The samples taken from lower test concentrations (0.298, 0.0931 and 0.0291 mg/L) were not analysed since these concentrations were not relevant for the determination of the toxicological endpoints.
- 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: Stock solutions were prepared by directly weighing 100, 31.3, 9.77 and 3.05 mg in 1000 mL test medium without algae, respectively and by directly weighing 1.91 mg in 2000 mL test medium without algae. These stock solutions were stirred in the dark at room temperature for 24 h (based on OECD Series on Testing and Assessment No. 23). Undissolved test item was allowed to sediment and/or float for two hours until the phases had separated. After the settling the necessary volumes for the test were withdrawn via a Teflon tube from the medium level of the stock solutions. The test item solutions for the three lowest test concentrations were made by diluting the next higher stock solution with test medium without algae to give the required test concentrations. Algae were added to each solution individually resulting in target cell densities of 0.5 × 10E+04 cells per mL in each solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel.
- Controls: blank control without test material
- Eluate: no
- Differential loading: no
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The stock solutions were clear and transparent after stirring but not the entire test item went into solution (oily droplets were observed). After the settling of the undissolved test material the necessary volumes for the test were withdrawn from the clear phase of the stock solutions.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green algae
- Strain: SAG 61.81
- Source: MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state.
- Conditions of cultivation: Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 μEm-2s-1); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.4 – 23.3 °C

pH:

Control: 7.44 – 7.99
Treatment: 7.26 - 7.99

Nominal and measured concentrations:

nominal: 100, 31.3, 9.77, 3.05, 0.954, 0.298, 0.0931, 0.0291 mg/L and control
measured: 0.652, 0.511, 0.343, 0.174, 0.0645 mg/L and control. The samples taken from lower test concentrations (0.298, 0.0931 and 0.0291 mg/L) were not analysed since these test concentrations were not relevant for the determination of the toxicological endpoints.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution
- Initial cells density: 0.5 × 10E+04 cells/mL (targeted)
- Control end cells density: 35.21 × 10E+04 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: AAP-Medium (according to Annex 3 of OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: Measurements of pH-value were performed at test start and end, the temperature was measured continuously and recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary
- Photoperiod: continuous light
- Light intensity and quality: 88.8 μEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0)). Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations in range finding study: Control, 100, 10.0, 1.00, 0.100 and 0.0100 mg/L
- Results used to determine the conditions for the definitive study: After 72 h at termination of the test a concentration response relation was observed for the inhibition of growth rate and yield from nominal test concentrations of 1.00 to 100 mg/L. The inhibition of growth rate peaked at 132.5 % and the inhibition of yield peaked at 100.6 % at a nominal test concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate; tested regularly

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.527 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.505 - 0.548 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.359 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.318 - 0.389 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.343 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.379 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.240 – 0.472 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.181 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.0376 – 0.269 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.174 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The cells were considered normal for the control and up to and including a nominal test concentration of 31.3 mg/L. No cells were observed at 100 mg/L test concentration.
For further details on biological results, please refer to section "Any other information on results incl. tables".

Results with reference substance (positive control):

EC50 (72 h, growth rate) = 0.971 mg/L
EC50 (72 h, yield) = 0.622 mg/L

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOELR and LOELR as well as NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided, for yield, Jonckheere Terpstra test, left sided, for growth rate). The EL10, 20, 50-values as well as EC10, 20, 50-values for growth rate and yield were determined by probit analysis following the Gompertz distribution. Due to statistical reasons inhibition-values below 0 % were set to zero. Only test concentrations within a clear concentration response relation were used for calculations.

Any other information on results incl. tables

Table 1: Average cell numbers for each sampling time and concentration

Nominal concentration	Average cell numbers [10^4/mL]
[mg/L]	0 h	24 h	48 h	72 h
Control	0.51	1.43	7.63	35.21
0.0291	0.51	1.73	7.78	32.46
0.0931	0.51	1.66	7.40	40.94
0.298	0.51	2.60	12.68	60.51
0.954	0.51	1.99	11.94	58.11
3.05	0.51	1.45	6.00	29.92
9.77	0.51	0.60	3.14	21.80
31.3	0.51	1.14	2.59	11.53
100	0.51	0.49	0.37	0.85

Table 2: Percentage inhibition of growth rate

Nominal concentration	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.0291	-21.1	-1.2	1.3^1)
0.0931	-18.5	1.6	-4.3^1)
0.298	-63.3	-19.6	-13.6^1)
0.954	-35.5	-17.2	-12.6^2)
3.05	-4.6	8.4	3.5
9.77	84.4	32.9	10.7
31.3	19.8	40.1	26.4*
100	103.5	118.3	93.9*

1) Value was omitted for EC10, 20, 50-calculation

2) Value was set to zero for EC10, 20, 50-calculation

* Statistically significant different to the control

Table 3: Percentage inhibition of yield

Nominal concentration	% Inhibition of yield
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.0291	-32.6	-2.3	7.9^1)
0.0931	-25.0	3.1	-16.5^1)
0.298	-127.2	-71.2	-72.9^1)
0.954	-60.9	-60.8	-66.0^2)
3.05	-2.2	22.8	15.2
9.77	90.2	63.0	38.6*
31.3	31.5	70.7	68.2*
100	102.2	102.0	99.0*

1) Value was omitted for EC10, 20, 50-calculation

2) Value was set to zero for EC10, 20, 50-calculation

* Statistically significant different to the control

Table 4: Main test: Individual cell numbers

Nominal concentration	Cell numbers [10^4/mL]				Yield
[mg/L]	0 h	24 h	48 h	72 h	0 h – 72 h
	0.52	2.18	9.67	45.66	45.14
	0.49	1.27	7.48	38.58	38.09
Control	0.52	1.23	6.91	30.54	30.02
	0.52	1.49	7.89	33.17	32.65
	0.49	1.10	8.17	40.92	40.43
	0.54	1.30	5.63	22.39	21.85
Mean	0.51^1)	1.43	7.63	35.21	34.60
	0.51	1.77	8.76	35.51	35.00
0.0291	0.51	2.05	8.07	32.83	32.32
	0.51	1.37	6.50	29.05	28.54
Mean	0.51	1.73	7.78	32.46	31.95
	0.51	1.70	4.93	40.85	40.34
0.0931	0.51	1.62	8.37	40.81	40.30
	0.51	1.67	8.89	41.16	40.65
Mean	0.51	1.66	7.40	40.94	40.43
	0.51	2.78	12.65	60.51	60.00
0.298	0.51	2.52	12.35	58.53	58.02
	0.51	2.51	13.04	62.49	61.98
Mean	0.51	2.60	12.68	60.51	60.00
	0.51	1.74	11.86	56.12	55.61
0.954	0.51	2.42	13.46	63.31	62.80
	0.51	1.82	10.50	54.91	54.40
Mean	0.51	1.99	11.94	58.11	57.60
	0.51	1.46	6.73	32.35	31.84
3.05	0.51	1.52	5.89	34.29	33.78
	0.51	1.37	5.38	23.13	22.62
Mean	0.51	1.45	6.00	29.92	29.41
	0.51	0.57	3.28	19.86	19.35
9.77	0.51	0.59	3.76	21.98	21.47
	0.51	0.63	2.39	23.55	23.04
Mean	0.51	0.60	3.14	21.80	21.29
	0.51	1.14	2.47	8.15	7.64
31.3	0.51	1.27	3.21	13.39	12.88
	0.51	1.01	2.08	13.05	12.54
Mean	0.51	1.14	2.59	11.53	11.02
	0.51	0.47	0.68	1.75	1.24
100	0.51	0.47	0.28	0.40	-0.11
	0.51	0.54	0.16	0.41	-0.10
Mean	0.51	0.49	0.37	0.85	0.34

1) The mean cell density of all control replicates is used as initial cell density for all treatment groups

Table 5: Determination of actual test item concentrations

Test item, nominal [mg/L]	Sampling	Test item, found			Actual test item concentration [mg/L]
		mg/L	% of nominal	Geometric mean [%]^1)
Control	0 h fresh	< LOD	-	-	-
	24 h aged	< LOD	-
	72 h aged	< LOD	-
0.954	0 h fresh	0.286	30	6.76	0.0645
	24 h aged	0.0883	9
	72 h aged	0.0191	2
3.05	0 h fresh	0.781	26	5.70	0.174
	24 h aged	0.235	8
	72 h aged	0.0521	2
9.77	0 h fresh	1.09	11	3.51	0.343
	24 h aged	0.503	5
	72 h aged	0.108	1
31.3	0 h fresh	1.31	4	1.63	0.511
	24 h aged	0.642	2
	72 h aged	0.226	1
100	0 h fresh	1.27	1	0.652	0.652
	24 h aged	0.729	1
	72 h aged	0.396	0.4

Table 6: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	factor of 69.04	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	26%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	6.7%; did not exceed 7% for the whole test period	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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