diethyl pyridine-2,4-dicarboxylate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 apr 2010 - 15 apr 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, Part C.11, 30 May 2008

Principles of method if other than guideline:

A limit test was performed based on the EU testing guidelines to demonstrate that the test item has no toxic effect on activated sludge up to the nominal concentration of 1000 mg/L.

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic: Inspection 2007-Nov-05 to 09 and 26 to 30, and 26 to 30-Nov-2007; decision 2008-Apr-30; signature: 2008-Nov-12

Test material

Test material information

Constituent 1

Reference substance name:

Mexoryl SBU

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

Mexoryl SBU

Constituent 2

Reference substance name:

diethyl pyridine-2,4-dicarboxylate

EC Number:

680-341-5

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

diethyl pyridine-2,4-dicarboxylate

Details on test material:

- Name of test material: MEXORYL SBU- The test material is the same than the one mentioned in section 6.1.3

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of three days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed* per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was 7.6.* Synthetic sewage feed:16g peptone11g meat extract 3g urea0.7g NaCl0.4g CaCl2 × 2H2O0.2g MgSO4 × 7H2O2.8g K2HPO4filled up to a final volume of 1 liter with deionized waterAt the start of the test, synthetic sewage feed and activated sludge inoculum were added. The inoculum had a sludge concentration of 2.7 g/L dry weight (corresponding to about 1.1 g dry material per liter test medium).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Test temperature:

20 °C

pH:

7.4 - 8.3

Dissolved oxygen:

8.2 - 9.0 mg/L

Nominal and measured concentrations:

1000 mg/L

Details on test conditions:

TEST SYSTEM- Test vessel: - Type: open - Material, size, headspace, fill volume: glass, 2000 mL, open, 500 mL - Aeration: continuously aerated by intense stirring on magnetic stirrers- No. of vessels per concentration (replicates): 1- No. of vessels per control (replicates): 2- Biomass loading rate: The inoculum had a sludge concentration of 2.7g/L dry weight (corresponding to about 1.1 g dry material per liter test medium)TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: local tap waterOTHER TEST CONDITIONSEFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration rate (= oxygen consumption rate); measured continually.For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. The oxygen consumption rate (in mg O2 L-1 minute-1) was determined from the linear part of the respiration curve in the range 6.5–2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. TEST CONCENTRATIONS- Limittest with 1000 mg/L- Justification for using less concentrations than requested by guideline: A limit test was performed based on the EU testing guidelines to demonstrate that the test item has no toxic effect on activated sludge up to the nominal test concentration of 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

>= 1 000 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:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 3

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

Effect concentrations 4

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

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? yes- The 3 hour EC50 of the reference item 3,5 dichlorophenol (positive control) was calculated to be 17 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons).- Relevant effect level: EC50 between 5 and 30 mg/L

Reported statistics and error estimates:

The 3 hour EC20, EC50 and EC80 values of the test item MEXORYL SBU and their 95% confidence limits could not be calculated because of the absence of a toxic effect.The 3 hour EC50 of the reference item 3,5 dichlorophenol was calculated by Probit Analysis.

Any other information on results incl. tables

Vessel no.	Nominal concentration of test chemical	Oxygen consumption rate	Inhibition	pH values		Oxygen concentration
						(mg O2/L)
	(mg/L)	(mg O2L^-1min^-1)	(%)	start*	end*	start*	end*
	Control
A	0	1.443		7.5	8.2	8.3	8.7
B	0	1.520		7.5	8.2	8.3	8.2
Mean		1.481
Deviation (%)		5.4
	Test item
5	1000	1.506	-1.7	7.4	8.1	8.2	8.8
	Reference item
1	5	1.547	-4.4	7.4	8.1	8.2	8.7
2	16	0.722	51.3	7.4	8.1	8.5	9.0
3	50	0.165	88.9	7.4	8.3	8.5	8.7

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The inhibitory effect of the test item MEXORYL SBU on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3‑hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of Chemicals, No. 209.

 

Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed.

 

In addition, two controls and three different concentrations of the reference item 3,5‑dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

 

The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at the test item concentration of 1000 mg/L.

 

Thus, the 3‑hour NOEC (EC₁₀) of the test item to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 1000 mg/L were not tested. The 3‑hour EC₂₀, EC₅₀, and EC₈₀could not be calculated but were clearly higher than 1000 mg/L.

The validity criteria were met.