1-(5-propyl-1,3-benzodioxol-2-yl)-1-ethanone

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:

05 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 209. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on 03-05 June 2013 / signed on 05 November 2013

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
See table 6.1.7/1: Dilution table for the test item
- Control: Test medium without test or reference item

Test organisms (species):

activated sludge

Details on inoculum:

- Source: Municipal sewage treatment plant of 31137 Hildesheim, Germany.
- Pretreatment: The sludge was washed twice with 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: 3.04 g/L corresponding to 1.52 g/L in the test vessel
- Dry solids concentration of sludge: 3.04 g/L; Dry solids concentration in test solution: 1.52 g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

21.7 °C

pH:

pH-value of the activated sludge 7.91
pH-value of the synthetic waste water 7.36

Dissolved oxygen:

Mean specific oxygen uptake rate of the control replicates was 20 mg 02/g·h

Salinity:

None

Nominal and measured concentrations:

3.2, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flasks, DIN 12380
- Aeration: Shaking of flasks at 150 rpm 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)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic waste water according to OECD Guideline 209
- Volume of the test medium: 500 mL
- Composition of test medium: The test item concentrations 3.2 mg/L was weighed out directly on object slides and placed into Erlenmeyer flasks. The further concentrations were pipetted directly in the Erlenmeyer flaks. See table 6.1.7/1

OTHER TEST CONDITIONS
- Adjustment of pH: Adjustment to 7.5 ± 0.5 was not necessary.

EFFECT PARAMETERS MEASURED
- 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.
- Physical Properties
pH: The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic wastewater was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
Oxygen: 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.
Temperature: The temperature was determined in the measuring cell once during the measurement. The room temperature during the test was recorded continuously by a hygrothermograph.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Preliminary test: 10, 100 and 1000 mg/L with two replicates each and without pH adjustment. Additionally, one preliminary test was carried out focusing on the inhibition of nitrification. 1000 mg/L were measured with and without addition of Allythiourea ATU (Final concentration: 11.6 mg/L). Additionally, one preliminary test was carried out focussing on the loss of test item during the incubation / aeration at 100 mg/L.
- Results used to determine the conditions for the definitive study: No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed. Based on these results the definitive study was carried out with 6 cocentrations within the range 3.2 - 1000 mg/L in a geometrical series and with a dilution factor of 3.2.

Reference substance (positive control):

yes

Remarks:

copper (II) sulphate pentahydrate

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

24.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CI 18.7-31.7 mg/L

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

48.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CI 39.5 - 58.7 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

189 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CI 165-219 mg/L

Key result

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

605 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CI 520 - 702 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The mean inhibition of total respiration for the test item replicates were -6, 6, 10, 35, 64 and 91% at 3.2, 10, 32, 100, 320 and 1000 mg/L, respectively.

Results with reference substance (positive control):

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 104 mg/L. This is in the recommended range of validity of 53 - 155 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 α-value (acceptable probability of incorrectly concluding that there is a difference) is α=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.

Table 6.1.7/2: Oxygen uptake rates and inhibition of the respiration of the control and test item Concentrationsafter 3 h Exposure Time (Inhibition of Total Respiration)

Test item concentration (mg/L)	Repl.	Oxygen Uptake Rate R (mg O2/L·h)	Inhibition (%)	Mean inhibition (%)
Control	1	32.4	-	-
	2	34.0
	3	31.2
	4	28.8
	5	26.4
	6	29.6
3.2	1	35.2	-16	-6
	2	29.6	3
	3	31.2	-3
	4	30.0	1
	5	34.8	-14
10	1	29.2	4	6
	2	28.4	7
	3	28.4	7
	4	28.8	5
	5	27.6	9
32	1	26.0	14	10
	2	27.6	9
	3	28.4	7
	4	26.4	13
	5	28.0	8
100	1	20.4	33	35
	2	20.0	34
	3	20.0	34
	4	18.8	38
	5	18.8	38
320	1	11.2	63	64
	2	10.8	64
	3	10.4	66
	4	11.6	62
	5	10.8	64
1000	1	1.6	95	91
	2	2.0	93
	3	3.2	89
	4	3.6	88
	5	2.8	91

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 8.9 %

Suspended solids in the test vessel: 1.52 g/L

Validity criteria:

- The mean specific oxygen uptake rate of the control replicates was 20 mg O2/g.h and therefore not meeting the criterion (validity criterion: should be ≥ 20 mg 02/g.h).

- The coefficient of variation of the oxygen uptake rates in the control replicates was 8.9% (validity criterion: < 30 %).

- The EC50 of the reference item was104 mg/L and in the required range of 53 - 155 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the NOEC is 10 mg/L. The EC10, EC20, EC50 and EC80 are 24.4, 48.7, 189 and 605 mg/L, respectively based on inhibition of total respiration.

Executive summary:

The effect of test item on the respiration rate of activated sludge was assessed according to OECD Guideline 209 with GLP statement.

 

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 3.2, 10, 32, 100, 320 and 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 mean inhibition of total respiration for the test item replicates were -6, 6, 10, 35, 64 and 91% at 3.2, 10, 32, 100, 320 and 1000 mg/L, respectively.

 

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 103 mg/L. This is in the recommended range of validity of 53 - 155 mg/L.

 

Under the test conditions, the NOEC is 10 mg/L. The EC10, EC20, EC50 and EC80 are 24.4, 48.7, 189 and 605 mg/L, respectively based on inhibition of total respiration.

Description of key information

OECD 209, GLP, key study, validity 1:

NOEC = 10 mg/L.

EC10 = 24.4 mg/L, based on inhibition of total respiration.

EC20 = 48.7 mg/L, based on inhibition of total respiration.

EC50 = 189 mg/L, based on inhibition of total respiration.

EC80 = 605 mg/L, based on inhibition of total respiration

Key value for chemical safety assessment

EC50 for microorganisms:

189 mg/L

EC10 or NOEC for microorganisms:

24 mg/L

Additional information

One key study is available (NOACK, 2014) to assess the toxicity of the registered substance to microorganisms. In this study, the effect of the registered substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours, according to OECD Guideline 209 with GLP statement.

 Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the tested substance concentrations 3.2, 10, 32, 100, 320 and 1000 mg/L. The respiration rates of the control, reference and tested substance 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 total respiration for the test item replicates were -6, 6, 10, 35, 64 and 91% at 3.2, 10, 32, 100, 320 and 1000 mg/L, respectively.

 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 103 mg/L. This is in the recommended range of validity of 53 - 155 mg/L.

 Under the test conditions, the NOEC is 10 mg/L. The EC10, EC20, EC50 and EC80 are 24.4, 48.7, 189 and 605 mg/L, respectively based on inhibition of total respiration.