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 microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 10 - 32 - 100 - 320 - 1000 mg/L additional 1000 mg/L with pH adjustment were weighed out directly in Erlenmeyer flasks
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 10 - 32 - 100 - 320 - 1000 mg/L
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non-adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.91 g/L, corresponding to 1.46g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.1 °C

pH:

pH-value of the activated sludge 7.96
pH-value of the synthetic waste water 7.43

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.



- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
- Composition of test medium:

Table for the Test Item Concentrations
Test Item Test concentration [mg/L]
10 32 100 320 1000 1000 pH
Test item [mg] 5 16 50 160 500 500
Inoculum [mL] 250
Synthetic waste water [mL] 16
Demineralised water [mL] filled up to 250 mL



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
Inhibition of Respiration (Preliminary Tests)

Inhibitions in the Preliminary Tests

Nominal Test Item Concentration [mg/L] pH(test item in demin. water) pH (adjusted to) pH (test item in medium) Inhibition [%]
Preliminary test – with pH adjustment
10 3.95 7.08* 7.37 14
3.80 - 7.34 15
100 2.52 7.98* 7.52 11
2.53 - 6.89 22 1000 1.63 6.18* 6.76 35
1.62 - 2.63 95

*)adjusted with 0.5 - -3.5 mol/L NaOH


Additional replicates were prepared to determine the inhibition of nitrification. Two replicates 1000 mg/L were measured with addition of Allylthiourea ATU (Final concentration: 11.6 mg/L). No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed.

Inhibitions in the Preliminary Test (with / without ATU)

Nominal Test Item Concentration Inhibition of total respiration Inhibition of heterotrophic respiration Inhibition of nitrification
[mg/L] [%] [%] [%]
1000 95 96 95

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

106 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 94.6 - 118 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

174 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 162 - 185 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

395 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 378 - 415 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

708 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 674 - 740 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 97.5 mg/L

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC 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 test are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O₂/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of the Control and Test Item Concentrations

	Test Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
	Control	1	33.2	–	–
		2	31.6
		3	31.6
		4	30.0
		5	30.0
		6	28.8
Test Item	10	1	31.2	-1	-2
		2	32.0	-4
		3	32.4	-5
		4	30.4	2
		5	31.6	-2
	32	1	31.6	-2	0
		2	30.0	3
		3	31.2	-1
		4	30.8	0
		5	30.8	0
	100	1	28.8	7	9
		2	27.2	12
		3	27.6	11
		4	28.4	8
		5	28.4	8
	320	1	18.8	39	41
		2	18.0	42
		3	18.4	40
		4	18.4	40
		5	18.0	42
	1000	1	0.8	97	98
		2	0.4	99
		3	0.8	97
		4	0.4	99
		5	0.4	99
	1000 pH	1	28.4	8	10
		2	28.8	7
		3	26.8	13
		4	26.8	13
		5	28.8	7

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 5.1%

Suspended solids in the test vessel: 1.46 g/L

Specific Respiration Rates of the Control Replicates

		Repl.	Specific Respiration Rate Rs [mg O2/g×h]
	Control	1	22.7
		2	21.6
		3	21.6
		4	20.5
		5	20.5
		6	19.7
Mean value			21.1

Repl. = Replicate

Suspended solids in the test vessel: 1.46 g/L

 

Oxygen Uptake Rates and Inhibition of the Respiration of the Reference Item Concentrations

	Test Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition   [%]	Mean Inhibition [%]
Reference Item	58	1	21.6	30	29
		2	22.0	29
		3	22.0	29
	100	1	15.2	51	48
		2	16.8	46
		3	16.0	48
	180	1	7.2	77	77
		2	7.2	77
		3	6.8	78

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the Test item is 32 mg/L.The test item is not toxic up to the concentration of 32 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 106 mg/L, the EC50 is 395 mg/L and EC80 is 708 mg/L of the test item.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item on 2015-01-09 in Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 - 320 - 1000 - 1000 pH mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -2 % to 98 %. Additionally, replicates with 1000 mg/L and pH-adjustment were prepared. The mean inhibition of the respiration of these replicates was 10 %.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 97.5 mg/L.

 

NOEC and EC-Values with Confidence Interval of the test item after 3 h exposure time, Inhibition of Total Respiration

	EC-values [mg/L]	Confidence interval P = 95 [%] [mg/L]
NOEC*	32	–
EC10	106	94.6 – 118
EC20	174	162 – 185
EC50	395	378 – 415
EC80	708	674 – 740

*) No statistically significant inhibition (P < 0.05), ANOVA, Dunnett`s Method

 

 

The NOEC of the test item is32mg/L. The test item is not toxic up to the concentration of 32 mg/L to activated sludge of a municipal sewage treatment plant. The EC10is 106 mg/L, the EC50is 395 mg/L and EC80is 708 mg/L of the test item.

Description of key information

The NOEC of the Test item is 32 mg/L.The test item is not toxic up to the concentration of 32 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 106 mg/L, the EC50 is 395 mg/L and EC80 is 708 mg/L of the test item.

Key value for chemical safety assessment

EC50 for microorganisms:

395 mg/L

EC10 or NOEC for microorganisms:

32 mg/L

Additional information

The NOEC of the Test item is 32 mg/L.The test item is not toxic up to the concentration of 32 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 106 mg/L, the EC50 is 395 mg/L and EC80 is 708 mg/L of the test item.