Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-, ammonium salt (1:1)

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:

25 January 2011 to 17 February 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Origin: municipal sewage treatment plant of D-31157 Sarstedt, Germany.
Reason for the selection: non adapted activated sludge from the sewage plant at Sarstedt is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.

The sludge was used whitin 24h after sampling.

Vehicle:

no

Details on test solutions:

TEST MEDIUM - Composition:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
test item concentration (directly weighted into each test vessel)

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Dry sludge concentration: 3.27g/L (corresponding to 1.64 g/L suspended solid in the test vessels).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

none

Hardness:

not reported

Test temperature:

18-22 °C
The temperature was determined in one control replicate prior to the oxygen measurement.
The room temperature during the test was recorded continuosly by a hygrothermograph.

pH:

The pH value of the activated sludge was determined prior to test start.
The pH value of the syntetic waste water was determine prior to the use and was adjusted to 7.5 +- 0.5.
The pH value of the test item was measured prior to incubation. Adjustment to 7.5 +- 0.5 was not necessary.

Dissolved oxygen:

The oxygen concentration was determined in each control, reference item and test item replicate after 3 hours incubation.
The oxygen concentration was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for 5 minutes on a flat bed recorder.

Salinity:

not reported

Nominal and measured concentrations:

Nominal concentration of the test item: 1000 mg/L
Nominal concentration of the reference substance (copper II) sulphanate pentahydrate: 180 - 100 - 58 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500mL Erlenmeyer flasks
- Aeration: permanent, to keep the dissolved oxygen concentration above 60-70% saturation and to maintain the sludge flakes in suspensione.
- Renewal rate of test solution (frequency/flow rate):



TEST ITEM
Concentration: 1000 mg/L
Replicates: 3
Vehicle: none
Dispersion treatment: Agitation

CONTROL (test medium without test or reference substance)
Replicates: 6 (triplicates at the beginning of the test + triplicatres at the end of the test)

REFERENCE ITEM (Copper (II) sulphanate pentahydrate)
purity: 99.8%
test concentration: 58 -100 - 180 mg/L
Replicates: single


OTHER TEST CONDITIONS
- Adjustment of pH:
The pH value of the activated sludge was determined prior to the test start.
The pH value of the synthetic waste water was determined prior to use and was adjusted to 7.5 +- 0.5.
The pH value of the test item replicates was measured prior to incubation. Adjustment to 7.5 +- 0.5 was not necessary.

- Photoperiod: n.a.
- Light intensity: n.a.


EFFECT PARAMETERS MEASURED: Oxygen uptake rate
After 3 hours 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 5 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: single concentration (1000 mg/L)
Based on the results of the preliminaty test the study was carried out with a limit test concentration.

- Range finding study
- Test concentrations: 10-100-1000 mg/L
- Results used to determine the conditions for the definitive study:
3% respiratory inhibition at 10 mg/L.
3% respiratory inhibition at 100 mg/L.
6% respiratory inhibition at 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Test item directly weighted into each vessel

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
none

- Effect concentrations exceeding solubility of substance in test medium:
it was not possible to determine an effect concentration.
The NOEC was determined to be >= 1000 mg/L.
The test item is higly soluble (solubility > 667 mg/L)

Results with reference substance (positive control):

- Results with reference substance valid?
yes

In the reference test the EC50-value for Copper (II) sulphate pentahydrate was determined by probit analysis to be:
EC50 = 72 mg/L
The reccomended range of validity of the reference substance is 53-155 mg/L, therefore the validity criteria was fullfilled.

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 Variancea Normality test and an Equal Variance test was done first. P values for both Normality and Equal Variance test are 0.05.
The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.005.

The EC50 -value of the reference item was calculated with probit analysis using software SigmaPlot (Windows), SPSS Corporation.
Probit value were taken from Weber (1986).

The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard method using EXCEL, MICROSOFT CORPORATION.

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

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

The results are listed in Table 1. The raw data are listed in Table 2.

Table 1
	Concentration (mg/L)	Oxygen Uptake Rate (mg O2 / L x h)	Specific Respiration Rate (mg O2 / g x h)	Inhibition %
test item 1	1000	28,30	17,3	5
test item 2	1000	28,10	17,1	5
test item 3	1000	27,40	16,7	8
control 1	-	30,00	18,3	-
control 2	-	29,80	18,2	-
control 3	-	30,20	18,4	-
control 4	-	25,20	15,4	-
control 5	-	30,50	18,6	-
control 6	-	32,60	19,9	-
Reference item	180	5,80	3,5	80
Reference item	100	10,10	6,2	66
Reference item	58	17,80	10,9	40
coefficient of variation of oxygen uptake rates of the control: 7.48%

 

 

Table 2
	Concentration (mg/L)	Oxygen concentration * (mg O2/L)	Measured distance ** (mm/5min)	Oxygen Uptake Rate (mg O2 / L x h)
test item 1	1000	6,61	118	28,30
test item 2	1000	5,57	117	28,10
test item 3	1000	6,46	114	27,40
control 1	-	6,90	125	30,00
control 2	-	6,99	124	29,80
control 3	-	6,74	126	30,20
control 4	-	7,37	105	25,20
control 5	-	7,63	127	30,50
control 6	-	7,64	136	32,60
Reference item	180	8,25	24	5,80
Reference item	100	7,90	42	10,10
Reference item	58	7,67	74	17,80
* determined at the start of the measurement
** 5 mm are related to 0.1 mgO2/L

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item cC6O4 ammonium salt is > 1000 mg/L.
cC6O4 ammonium salt caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

Executive summary:

A Respiratory Inhibition Test with activated sludge according to OECD guideline No. 209 was carried out with the test item cC6O4 ammonium salt (batch number: 156/17).

Test system was activated sludge of the municipal treatment plant of D-31157 Sarstedt. The test system was carried out under static conditions with limit concentration 1000 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 inhibition of respiration for the test item replicates ranged from 5 % to 8 %.

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 72 mg/L. Validity criteria of the test guideline were fulfilled.

The NOEC of the test item cC6O4 ammonium salt is > 1000 mg/L.

cC6O4 ammonium salt caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

Description of key information

A 3 hours Respiratory Inhibition Test with activated sludge according to OECD guideline No. 209 was carried out with the test item cC6O4 ammonium salt. The test system was carried out under static conditions with limit concentration 1000 mg/L. The study was performed under GLP without deviations.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

cC6O4 ammonium salt caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

NOEC > 1000 mg/L.