Reaction mass of diisopropyl hydrogen phosphate and {[hydroxy(propan-2-yloxy)phosphoryl]oxy}(propan-2-yloxy)phosphinic acid and isopropyl dihydrogen phosphate and methyl dihydrogen phosphate and phosphoric acid

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-16 to 2015-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels (pH-neutralised and un-neutralised) and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (48 hours). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000). Details of the analytical method are presented
in section 13. Analytical results are presented in section 8.2.2.

Sampling for the analytical monitoring
At the start of the exposure (0 hours), sampling was carried out after preparation of the test concentrations.
At the end of the exposure (48 h), samples were taken directly from the test vessels.

Quality criteria for the analytical monitoring
Recoveries of the test item should be within ± 20 % of the nominal or initially measured concentrations. The effect levels can be given based on the nominal
concentrations, if this quality criteria is fulfilled

Vehicle:

no

Details on test solutions:

Stock solution
The stock solution (100 mg/L of the test item were weighed out) was freshly prepared in a measuring flask with dilution water (see below) before the start of the exposure phase (at 0 hours).

Treatment
Agitation

Test concentrations /solutions
5 test item concentrations in a geometric series with a separation
factor of 2, prepared by diluting the stock solution of 100 mg/L with dilution water, were tested as follows:
6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.
The test concentrations were selected based on the results of a non GLP preliminary range finding test. For results, see section 8.1.

pH-neutralised concentration
The highest concentration level of 100 mg/L was initially adjusted to a pH in the range of 7.0 ± 0.2 by addition of 1 M NaOH and tested under the same
conditions as the un-neutralised concentration levels.

Control
Dilution water without test item tested under the same conditions as the test groups

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Clone 5
- Source:
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Age at study initiation (mean and range, SD): 2 to 24 h old daphnids from a healthy stock were used for the study
- Method of breeding:
Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +- 2°C, in an incubator, 16 h illumination; light intensity
of max. 20 µE x m-2 x s-1
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Cultur feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and
Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: At least 2 h in dilution water.
- Acclimation conditions (same as test or not): Same as test conditions.
- Health during acclimation (any mortality observed): No mortality observed.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2015-06-16 (0h) 262 mg/L CaCO3

Test temperature:

18 - 22°C, constant within ± 1°C
2015-06-16 20.4°C

pH:

Water Quality Parameters at the start of the Exposure (0 hours)

Nominal
test item
concentrations Dissolved
[mg/L] pH-value O2-concentration

100 (pH neutralised*) 6.96 8.60
100 (un-neutralised) 5.18 8.61
50.0 6.44 8.50
25.0 6.95 8.60
12.5 7.29 8.57
6.25 7.61 8.54
Control 7.96 8.86


Water Quality Parameters at the End of the Exposure (48 hours)

Nominal
test item
concentrations
[mg/L] pH-values Dissolved O2 -concentration [mg/L] Replicates Replicates
1 2 3 4 1 2 3 4
100 (pH neutralised*) 7.22 7.35 7.36 7.37 8.22 8.08 7.95 8.01
100 (un-neutralised) 6.11 5.82 5.74 5.68 8.35 7.93 8.35 8.34
50.0 7.29 7.26 7.24 7.27 8.27 8.23 8.02 8.22
25.0 7.58 7.58 7.56 7.60 8.11 8.15 8.15 8.35
12.5 7.67 7.66 7.65 7.63 8.14 8.10 7.90 7.87
6.25 7.77 7.74 7.70 7.67 8.35 8.23 7.95 7.62
Control 7.90 7.92 7.94 7.95 8.24 8.54 8.63 8.63
* = pH-value adjusted from pH4.33 to pH 7.12 by addition of 1 M NaOH

Dissolved oxygen:

Water Quality Parameters at the Start of the Exposure (0 hours)

Nominal
test item
concentrations Dissolved
[mg/L] pH-value O2-concentration

100 (pH neutralised*) 6.96 8.60
100 (un-neutralised) 5.18 8.61
50.0 6.44 8.50
25.0 6.95 8.60
12.5 7.29 8.57
6.25 7.61 8.54
Control 7.96 8.86

Water Quality Parameters at the End of the Exposure (48 hours)

Nominal
test item
concentrations
[mg/L] pH-values Dissolved O2 -concentration [mg/L] Replicates Replicates
1 2 3 4 1 2 3 4
100 (pH neutralised*) 7.22 7.35 7.36 7.37 8.22 8.08 7.95 8.01
100 (un-neutralised) 6.11 5.82 5.74 5.68 8.35 7.93 8.35 8.34
50.0 7.29 7.26 7.24 7.27 8.27 8.23 8.02 8.22
25.0 7.58 7.58 7.56 7.60 8.11 8.15 8.15 8.35
12.5 7.67 7.66 7.65 7.63 8.14 8.10 7.90 7.87
6.25 7.77 7.74 7.70 7.67 8.35 8.23 7.95 7.62
Control 7.90 7.92 7.94 7.95 8.24 8.54 8.63 8.63
* = pH-value adjusted from pH4.33 to pH 7.12 by addition of 1 M NaOH

Nominal and measured concentrations:

The concentrations of the test item Hordaphos CC MIS were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and at the end of the exposure (48 hours) in all concentration levels (pH-neutralised and un-neutralised) and the control. Details of the analytical method are presented in section 13.
The measured concentrations of Hordaphos CC MIS at the start of the exposure (0 hours) were in the range of 99 to 105 % of the nominal values. At the end of the exposure (48 hours), the measured concentrations were in the range of 97 to 102 % of the nominal values.
The measured concentrations of the test item Hordaphos CC MIS were all within ± 20 % of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 cm ID x 7 cm H), 50 mL capacity, loosely covered with watch glasses
- Test volume: 20 mL
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.75
NaHCO3 64.8
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123

- Water Quality Parameters of the Dilution Water prior to the Start of the Exposure (0 h):
pH-value Dissolved O2 concentration [mg/L] Temperature [°C] Conductivity [µS/cm] Total hardness [mg CaCO3/L]
0h 7.96 8.86 20.4 661 262

- Culture medium different from test medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: test start (0 h) and test end (48 h)

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE x m-2 x s-1 (<1340 lx)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation, 24 h interval

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

See below

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 hours. The EC50-value with 95 % confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 2.00 mg/L (Cl 1.76 - 2.27 mg/L)
The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Reported statistics and error estimates:

Methods of evaluation
The EC-values (after 24 and 48 hours) as well as the NOEC and LOEC (after 48 hours) were deduced directly from the observation data, without mathematical calculation. Since the measured concentrations of the test item Hordaphos CC MIS were all within ± 20 % of the nominal concentrations, all effect levels (EC10 / 50 / 100, NOEC and LOEC) were based on the analytically confirmed nominal concentrations of the test item.

EC-values and statistical analyses

The EC50-value for the reference item was calculated by sigmoidal
dose-response regression. The respective 95 % confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5.

Software
All data were computer processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations
based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Biological Data

 

The percentage immobility, determined in all test and control groups after 24 and 48 hoursunder static conditions, is given.

Immobilisation Rates after 24 and 48 h ofExposure in the Definitive Test  (n = 20, divided into 4 replicates with 5 daphnids each)

HordaphosCC MIS	IMMOBILISATION [%]
Nominal test item concentration [mg/L]	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100 (pHneutralised*)	0	0	0	0	0	0	0	0	0	0
100 (un-neutralised)	0	0	0	0	0	0	0	0	0	0
50.0	0	0	0	0	0	0	0	0	0	0
25.0	0	0	0	0	0	0	0	0	0	0
12.5	0	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

*    = pH-value adjusted from pH 5.18 to pH 6.96 by addition of 1 M NaOH

 

All test concentrations were visually clear throughout the exposure period.

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

No effects on Daphnia magna were observed at any of the tested pH-neutralised and un-neutralised concentration levels of 6.25 to 100 mg/L of the test item Hordaphos CC MIS and a pH-range of 5.18 to 7.77. Thus, the observed effects at the un-neutralised concentration level of 100 mg/L in the non GLP preliminary range finding test were induced by a pH-value of ≤ 4.63

Executive summary:

In the acute immobilisation test withDaphnia magna(STRAUS), the effects of the test item Hordaphos CC MIS (batch number:DEH2 035729) were determined according to OECD 202 (2004) from2015-06-16 to 2015-06-19, with the definitive exposure phase from 2015-06-16 to 2015-06-18,at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The study was conducted under static conditions over a period of 48 hours with 5 concentrations of the test item Hordaphos CC MIS in the range of 6.25 to 100 mg/L, prepared with dilution water in a geometric series with a separation factor of 2. Additionally, the highest test concentration of 100 mg/L was tested pH-neutralised.The pH neutralisation reflects the actually existing environmental conditions in the buffered aquatic systems. The test item concentrations were selected based on the results of a non GLP preliminary range finding test, showing significant effects at an un-neutralised concentration of 100 mg/L.

The test itemHordaphos CC MISis a colourless, clear, viscous liquid, which is miscible with water, and is known to cause a low pH value in aqueous solution at higher concentrations (e.g. pH 1.3 at 10 g/L, 20 °C).

A stock solution was freshly prepared before the start of the exposure period. The stock solution of 100 mg/L of the test item was homogenised by manual agitation and used as highest test concentration and for preparation of the concentration levels of 6.25 to 50.0 mg/L. Additionally, the test concentration of 100 mg/L wasadjusted to a pH in the range of 7.0±0.2 by addition of 1 M NaOH and tested under the same conditions as the un-neutralised concentration levels.The tested solutions were visually clear throughout the exposure period.

Twenty daphnids were exposed to each concentration level and the control.

The concentrations of the test item Hordaphos CC MIS were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and at the end of the exposure (48 hours) in all concentration levels (pH-neutralised and un-neutralised) and the control.

The measured concentrations at the start of the exposure (0 hours) were in the range of 99 to 105 % of the nominal values. At the end of the exposure (48 hours), the measured concentrations were in the range of 97 to 102 % of the nominal values.

The measured concentrations of the test item Hordaphos CC MIS were all within ± 20 % of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test. The effect levels given inTable1are based on the nominal concentrations of the test item Hordaphos CC MIS.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 hours) and at the end of the exposure (48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

 

Description of key information

No effects on Daphnia magna were observed at any of the tested pH-neutralised and un-neutralised concentration levels of 6.25 to 100 mg/L of the test item Hordaphos CC MIS and a pH-range of 5.18 to 7.77. Thus, the observed effects at the un-neutralised concentration level of 100 mg/L in the non GLP preliminary range finding test were induced by a pH-value of ≤ 4.63