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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-23 to 2015-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All concentration levels, the pH control and the control were analytically verified via LC-MS/MS after 0 hours (start of exposure) and 72 hours (end of exposure). Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates.

Vehicle:

no

Details on test solutions:

Stock solution
100 mg test item/L, freshly prepared with dilution water

Media preparation
Direct addition of the test item and shaking until the test item is completely dissolved.

Test concentrations
5 concentrations were tested in a geometrical series with a (nominal) dilution factor of ∜10=1.78: 10.0 - 17.8 - 31.7 - 56.4 - 100 mg/L.
Three replicates of the highest test concentration, adjusted with 1M NaOH and HCl to pH 8.1 ± 0.2, were tested under the same test conditions as the test vessels. The concentrations were based on the results of a preliminary range finding test (non-GLP).

Control
Six replicates (without test item) were exposed under the same conditions as the test concentrations.

Test organisms (species):

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

Details on test organisms:

Test organism
Pseudokirchneriella subcapitata HINDÁK, SAG 61.81

Reason for the selection of the test organism
Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin Sammlung von Algenkulturen (SAG)
Pflanzenphysiologisches Institut der Universität Göttingen
Nikolausberger Weg 18, D-37073 Göttingen

Cultivation at test facility
Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE•m-2•s-1 for 24 hours per day.

Culture medium
Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 21.5 max.: 23.0, mean value: 22.3

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
pH-Control 8.23 7.80
100 3.60 3.42
56.4 4.76 4.39
31.7 7.15 5.91
17.8 7.59 6.27
10.0 7.80 7.13
Control 8.19 8.24

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

nominal: 10.0 - 17. 8 - 31.7 - 56.4 - 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 6153 cells/mL
- Control end cells density: Mean 2510377 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 67.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:

Biological Data

The results of the range finding test are presented below:

Results of the Range Finding Test (0 - 72 hours)
Nominal Test Item Concentration [mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 114 100
10.0 -3 -15
1.00 -4 -26
pH-Control (100 mg/L, adjusted to 8.1) 6 30
Negative values = growth stimulation

Analytical Data

The analytical data of the range finding test are presented below:

Measured Exposure Concentrations during the non GLP Preliminary Range Finding Test
Determination of the test item via LC-MS/MS, with a preliminary non validated analytical method

Sampling time 0 h 72 h
Test item Meas. Meas. Recovery
concentration conc. Recovery conc. [%]
[mg/L] [mg/L] [%] [mg/L]

10.0 9.80 98 10.5 105
Meas. conc = Measured concentration of the test item, dilution factor taken into account

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

27.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 23.0 - 30.9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 27.3 - 31.7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

35.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 32.8 - 44.3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 19.3 - 22.6

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The toxicity of potassium dichromate (SIGMA, batch number MKBQ9179V, purity 100 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2015-04-14 to 2015-04-17 (with headspace). The reference item toxicity is in the valid range following test facility SOPS. For results, see Table 10.

EC50-Values of the Reference Item based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.613 0.810 ± 0.404
95 % confidence interval 0.589 - 0.638
Yield inhibition
EyC50 0.281 0.421 ± 0.274
95 % confidence interval 0.234 - 0.314
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Cell Densities

Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours		48 hours		72 hours
100 mg/L pH-Control adjusted to pH 8.1		1		6153		31733		393211		2270075
		2		6153		42389		400549		2226924
		3		6153		32436		449345		2173612
		Mean		6153		35519		414368		2223537
100		1		6153		<LOQ		3409		4340
		2		6153		3588		3644		<LOQ
		3		6153		3033		<LOQ		<LOQ
		Mean		6153		n.a.		n.a.		n.a.
56.4		1		6153		3534		<LOQ		<LOQ
		2		6153		3525		4055		<LOQ
		3		6153		3754		3776		3503
		Mean		6153		3604		n.a.		n.a.
31.7		1		6153		43123		414504		681252
		2		6153		39100		328358		632299
		3		6153		41270		347895		673381
		Mean		6153		41164		363586		662311
17.8		1		6153		49138		552927		1358064
		2		6153		43929		401803		1459168
		3		6153		35412		319797		1459796
		Mean		6153		42826		424842		1425676
10.0		1		6153		46984		423881		1767375
		2		6153		33969		355328		1994609
		3		6153		33496		339805		1772518
		Mean		6153		38150		373005		1844834
Control		1		6153		55034		605518		2615349
		2		6153		51895		538658		2602742
		3		6153		47294		464178		2294850
		4		6153		45127		442540		2902983
		5		6153		33737		375461		2095526
		6		6153		45767		427581		2550810
		Mean		6153		46476		475656		2510377

< LOQ = below the limit of quantification (2736 cells/mL)                            n.a. = data not determinable

 

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Nominal test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100 mg/L pH-Control adjusted to pH 8.1	1		1.97	2		2263922	10
	2		1.96	2		2220771	11
	3		1.96	2		2167459	13
	Mean	(-)	1.96	2	(-)	2217384	11
100	1		n.a.	100		n.a	100
	2		n.a	100		n.a	100
	3		n.a	100		n.a	100
	Mean	(+)	n.a	100	(+)	n.a	100
56.4	1		n.a	100		n.a	100
	2		n.a	100		n.a	100
	3		n.a	100		n.a	100
	Mean	(+)	n.a	100	(+)	n.a	100
31.7	1		1.57	22		675099	73
	2		1.54	23		626146	75
	3		1.57	22		667228	73
	Mean	(+)	1.56	22	(+)	656158	74
17.8	1		1.80	10		1351911	46
	2		1.82	9		1453015	42
	3		1.82	9		1453643	42
	Mean	(+)	1.82	9	(+)	1419523	43
10.0	1		1.89	6		1761222	30
	2		1.93	4		1988456	21
	3		1.89	6		1766365	29
	Mean	(+)	1.90	5	(+)	1838681	27
Control	1		2.02			2609196
	2		2.02			2596589
	3		1.97			2288697
	4		2.05			2896830
	5		1.94			2089373
	6		2.01			2544657
	Mean		2.00			2504224

Validity criteria fulfilled:

yes

Conclusions:

In this study, Hordaphos CC MIS was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours when tested with 100 mg/L and a pH adjusted to 8.1. The NOEC (0-72 h) for both inhibition of growth and inhibition of yield was 100 mg/L.As the toxic effects are due to the pH value this was additionally investigated in a geometrical series of five test concentrations. Under these conditions, the following effect values were determined: The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 35.3 (32.8 – 44.3) mg/L and 20.9 (19.3 – 22.6) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were < 10.0 mg/L. All effect values are based on the nominal concentrations of Hordaphos CC MIS.

Executive summary:

The toxicity of Hordaphos CC MIS (Batch no.: DEH2 035729) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 761/2009/Method C.3 from 2015-06-23 to 2015-06-26 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany. The aim of the study was the determination of NOEC, LOEC, EC₁₀- , EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A stock solution of 100 mg/L was freshly prepared with dilution water. In the range finding test, it was observed that the toxic effect of Hordaphos CC MIS is due to the pH of the test solutions. Therefore, a limit test concentration of 100 mg/L was tested with a pH value adjusted to 8.1. Additionally, the influence of the pH value was investigated in a geometrical series of five test concentrations with a dilution factor of (nominal):10.0 - 17. 8 - 31.7 - 56.4 - 100 mg/L. The environmental conditions were within the acceptable limits.

 

The test media were visually clear throughout the test period. The concentrations of the test item Hordaphos CC MIS in all test concentrations and the control were analytically verified by LC-MS/MS at the start and the end of exposure.
The measured concentrations of Hordaphos CC MIS in the fresh media (0 h) were in the range of 97 to 112 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 106 to 110 % of the nominal values of all concentrations.

Therefore, all effect values given are based on the nominal test item concentrations of Hordaphos CC MIS.

 

The two highest concentrations caused a change of the pH value to be below 5. The toxic effects are due to the pH value. Replicates with the test item concentration of 100 mg/L adjusted to pH 8.1 showed no toxic effects.

 

NOEC, LOEC, EC-values and 95 % Confidence Intervals of HordaphosCC MIS (0 - 72 hours)

                  based on nominal test item concentrations [mg/L]

	Not pH-Neutralized	pH-Neutralized
	Growth RateInhibition	Growth RateInhibition
NOEC	< 10.0	100
LOEC	10.0	> 100
ErC10	27.8 (23.0 – 30.9)	> 100
ErC20	31.3 (27.3 – 31.7)	> 100
ErC50	35.3 (32.8 – 44.3)	> 100
	Inhibition of Yield	Inhibition of Yield
NOEC	< 10.0	< 100
LOEC	10.0	100
EyC10	< 10.0	< 100
EyC20	< 10.0	> 100
EyC50	20.9 (19.3 – 22.6)	> 100

 

Description of key information

In this study, Hordaphos CC MIS was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours when tested with 100 mg/L and a pH adjusted to 8.1. The NOEC (0-72 h) for both inhibition of growth and inhibition of yield was 100 mg/L.

As the toxic effects are due to the pH value this was additionally investigated in a geometrical series of five test concentrations. Under these conditions, the following effect values were determined: The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 35.3 (32.8 – 44.3) mg/L and 20.9 (19.3 – 22.6) mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were < 10.0 mg/L. All effect values are based on the nominal concentrations of Hordaphos CC MIS.