Dihydro-3-(tripropenyl)furan-2,5-dione

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-24 to 2012-09-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study, no deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tripropenyl succinic acid

Cas Number:

147384-84-7

Molecular formula:

R-C4H6O4 , whereas R=C8-C10-alkyl-(branched, unsaturated)

IUPAC Name:

Tripropenyl succinic acid

Details on test material:

- Name of test material (as cited in study report): Tripropenyl succinic acid
- Physical state: Viscous liquid, pale brown
- Analytical purity: 99.3 %
- Purity test date: 2013-03-04
- Lot/batch No.: 12-SK002
- Expiration date of the lot/batch: January 2014
- Stability under test conditions: Not applicable
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was weighed out directly in an Erlenmeyer flask and filled up with 200 mL demineralised water. Prior to addition of the activated sludge, the pH value was adjusted to 7 +/- 0.5.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 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

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: Washed twice with autoclaved tap water and was used within 24 h after sampling. Adjustment to a dry sludge concentration of 3.0 g/L +/- 10 % was necessary.
- Dry sludge concentration: 2.76 g/L

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

Not determined

Test temperature:

21.6 °C

pH:

pH-value of the activated sludge 7.32
pH-value of the synthetic waste water 7.45

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask
- Aeration: Shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain
the sludge flakes suspension.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
Test item (directly weighing)

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 Test)

Nominal Test Item Concentration Pretreatment pH Inhibition [%]
[mg/L] (test item in water)
1 None - 1
10 4
100 - 50
1000 - 99
1000 pH adjusted 9.70 (7.03*) 80
1 Stirring overnight - 2
10 - 2
100 - 11
1000 - 97
1000 pH adjusted 3.22 (6.92*) 17

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

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

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 116 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, if necessary, 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 a-value (acceptable probability of incorrectly concluding that there was a difference) is a=0.05.

The EC-values of the test item were calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPad Prism. Calculations of the confidence intervals for the EC50-values were carried out using standard procedures.

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

Any other information on results incl. tables

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 Reference Item Concentrations

Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	22.8	16.5	–	–
	2	23.2	16.8
	3	22.0	15.9
	4	22.8	16.5
	5	26.4	19.1
	6	26.8	19.4
58	1	12.8		(47)*	4
	2	23.6		2
	3	22.8		5
100	1	14.8		38	40
	2	15.2		37
	3	13.2		45
180	1	5.6		77	80
	2	5.2		78
	3	3.6		85

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

Test item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
10	1	22.0	8	7
	2	22.0	8
	3	22.8	5
	4	22.8	5
	5	22.0	8
32	1	22.8	5	6
	2	23.2	3
	3	22.8	5
	4	22.8	5
	5	21.2	12
100	1	22.4	7	7
	2	22.4	7
	3	22.4	7
	4	22.4	7
	5	22.4	7
320	1	19.6	18	16
	2	20.8	13
	3	20.4	15
	4	20.4	15
	5	19.6	18
1000	1	18.0	25	33
	2	17.6	27
	3	16.4	32
	4	16.0	33
	5	12.8	47

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of the test item is > 1000 mg /L and the EC20 is 378 mg/L. The test item is not toxic up to the concentration of 100 mg/L to activated sludge of a municipal sewage treatment plant. The NOEC of the test item is 100 mg/L.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Tripropenyl succinic acid (batch number: 12-SK002) from 2012-09-24 to 2012-09-27, with the experimental phase on 2012-09-27 at Dr.U.Noack-Laboratorien, D-31157 Sarstedt. Test system was activated sludge of the municipal treatment plant of D-31137 Hildesheim. The test was carried out under static conditions with the nominal test item concentrations 10 - 32 - 100 - 320 - 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 respiration for the test item replicates ranged from 6 % to 33 %.

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 116 mg/L.

NOEC and EC-Values with Confidence Interval of Tripropenyl succinic acid

		EC-values		Confidence interval
		[mg/L]		P = 95 [%] [mg/L]
NOEC^*		100		–
EC20		378		324 - 443
EC50		> 1000		n.d.
EC80		> 1000		n.d.

                                                           *) No statistically significant effect (p 0.05), ANOVA and Dunnett’s Method

                                               n.d. = not determinable

The EC50 of the test item is > 1000 mg /L and the EC20 is 378 mg/L. The test item is not toxic up to the concentration of 100 mg/L to activated sludge of a municipal sewage treatment plant. The NOEC of the test item is 100  mg/L.