Dimethyl (p-methoxybenzylidene)malonate

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

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: 3.2 - 10 - 32 - 100 - 320 - 1000 mg/L . 3.2, 10 and 32 mg/L were weighed out onto object slides and transferred with these into the Erlenmeyer flasks. The other concentrations were weighed out directly in the Erlenmeyer flasks. 200 mL demineralised water was added and stirred overnight.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 3.2 mg/L; 10 mg/L; 32 mg/L; 100 mg/L; 320 mg/L; 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
-Reference item: three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of 1.8., Triplicates

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)
- Receipt: 2015-12-07
- 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: 2.78 g/L, corresponding to 1.39 g/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:

21.7 °C

pH:

pH-value of the activated sludge: 7.29
pH-value of the synthetic waste water: 7.27

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.
- Pretreatment: Stirring overnight

- 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.)
- No. of vessels per reference item (replicates): 9 (three concentrations within the range 58 - 180 mg/L and a dilution factor 1.8,at the end of the test)
- Composition of test medium:

Dilution Table for the Test Item
Test item concentration [mg/L] 3.2 10 32 100 320 1000
Test item [mg] 1.6 5 16 50 160 500
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250


Dilution Table for the Reference Item
Reference item concentration [mg/L] 58 100 180
Stock solution [µL]* 96.7 166.7 300
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
* Stock solution with 300g/L


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
A preliminary test (non GLP) was carried out with the concentrations of the test item Hostavin PR-25 P of 10 – 100 – 1000 mg/L with two replicates each. The flasks were aerated for 3 hours. The first replicates of each concentration were pH adjusted.

Inhibitory effects of the test item do not rely on pH effects, therefore, no pH adjustment is necessary.

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


[%]
1 st Preliminary test – with pH adjustment
10 5.58 6.30* 7.24 8
5.62 - 7.19 10
100 5.44 6.57* 7.13 39
5.72 - 7.22 38
1000 5.51 6.71* 7.25 41
5.51 - 7.22 47
*) adjusted with 0.05 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
[mg/L] Inhibition of total respiration
[%] Inhibition of heterotrophic respiration
[%] Inhibition of nitrification
[%]
1000 41* 25* 58*
47 33 60
*) measured from pH-adjusted replicates

Based on these results the definitive study was carried out with 6 concentrations within the range of 3.2 - 1000 mg/L in a geometrical series and with a dilution factor of 3.2. The test item was weighed out onto object slides and transferred with these into the Erlenmeyer flasks or directly weighed out in Erlenmeyer flasks, respectively.

A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice. Adjustment to a dry sludge concentration of 3.0 g/L ± 10 % was done by dilution with tap water (actual dry sludge concentration 2.78 g/L).

The oxygen uptake rates of all samples were determined following an incubation period of 3 h.

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

33.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 29.7 - 37.8 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

68.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 60.3 - 78.7 mg/L

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

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC10: 33.7 mg/L; EC20: 68.5 mg/L; EC50: >1000 mg/L; EC80: >1000 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 alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=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	30.4	–	–
		2	29.6
		3	28.4
		4	26.4
		5	25.6
		6	26.0
Test Item	3.2	1	28.4	-3	-7
		2	30.0	-8
		3	29.2	-5
		4	30.0	-8
		5	30.4	-10
	10	1	27.2	2	-2
		2	28.0	-1
		3	28.4	-3
		4	28.4	-3
		5	28.4	-3
	32	1	25.6	8	9
		2	24.8	10
		3	26.0	6
		4	24.8	10
		5	25.2	9
	100	1	20.4	26	25
		2	21.2	23
		3	21.6	22
		4	20.8	25
		5	19.6	29
	320	1	19.6	29	31
		2	18.8	32
		3	18.8	32
		4	18.8	32
		5	18.8	32
	1000	1	18.8	32	34
		2	18.8	32
		3	18.0	35
		4	18.4	34
		5	18.0	35

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 7.3%

Suspended solids in the test vessel: 1.39 g/L

Specific Respiration Rates of the Control Replicates

		Repl.	Specific Respiration Rate Rs [mg O2/g×h]
	Control	1	21.9
		2	21.3
		3	20.4
		4	19.0
		5	18.4
		6	18.7
Mean value			20.0

Repl. = Replicate

Suspended solids in the test vessel: 1.39 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	20.8	25	25
		2	20.8	25
		3	20.8	25
	100	1	15.6	44	47
		2	14.8	47
		3	14.0	49
	180	1	6.8	75	76
		2	6.8	75
		3	6.4	77

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item is 10 mg/L. The test item is not toxic up to the concentration of 10 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 33.7 mg/L and the EC20 is 68.5 mg/L. The EC50 and EC80 are > 1000 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 from 2015-12-07 to 2015-12-08, with the definitive exposure phase on 2015-12-08 at the test facility. 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 3.2 - 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 -7% to 34%.

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.

The NOEC of the test item is 10mg/L. The test item is not toxic up to the concentration of 10 mg/L to activated sludge of a municipal sewage treatment plant. The EC10 is 33.7 mg/L and the EC20 is 68.5 mg/L. The EC50 and EC80 are > 1000 mg/L of the test item.

 

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*	10	–
EC10	33.7	29.7 – 37.8
EC20	68.5	60.3 – 78.7
EC50	> 1000	–
EC80	> 1000	–

*) No statistically significant inhibition (P < 0.001), ANOVA, Dunnett`s Method

Description of key information

3h-EC10 (activated sludge, total respiration inhibition) = 33.7 mg/L, 3h-EC50 (activated sludge, total respiration inhibition) > 1000 mg/L (OECD 209, 2016)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

33.7 mg/L

Additional information

In a reliable key study, respiration inhibition of the submission substance on activated sludge was investigated according to OECD 209 and in compliance with GLP (RL1). The test was carried out with five test concentrations (3.2 - 10 - 32 - 100 - 320 - 1000 mg/L) under static conditions for three hours. Controls were run in parallel. Inhibition of total respiration followed a dose-response relationship. The submission substance was not toxic up to a concentration of >= 10 mg/L. The determined NOEC was therefore 10 mg/L. Further calculated effect concentrations were (nominal): EC10 = 33.7 mg/L, EC20 = 68.5 mg/L, EC50 / EC80 > 1000 mg/L. The study was considered reliable and adequate for the environmental hazard assessment for STP microorganisms.