diethyl pyridine-2,4-dicarboxylate

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 apr 2010 - 29 jul 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. The study report was conclusive, done to a valid guideline and the study was conducted 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 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Directive 92/69/EEC C.4-C, 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

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:

Identity:R0037857ACAS No.41438-38-4Batch No.:022D001Purity:100%Carbon Content % (w/w):59.19%Expiration Date:31-Oct-2012Aggregate State / Physical Form at Room Temperature:Crystallized solid which can be liquid depending on the room temperature (low melting point 30°C)Color:YellowSolubility in Water:~10 g/LStorage Conditions:At room temperature, away from light and shielded from humidity.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

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, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was diluted with test water to a concentration of about 1 g dry material per liter. Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

25.6 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

15.2 other: mg TOC/L

Based on:

other: the percentage carbon content of the test item (59.2%) provided by the Sponsor

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST WATERThe test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stocksolutions:1)KH2PO48.50 g/LK2HPO421.75 g/LNa2HPO4 × 2H2O33.40 g/LNH4Cl0.50 g/LThe pH of this solution was 7.4.2)MgSO4 × 7H2O22.50 g/L3)CaCl2 × 2H2O36.40 g/L4)FeCl3 × 6H2O0.25 g/L, stabilized with one drop of concentrated HCl per literTo obtain the final test water, 10 mL of stock solution 1) and 1 mL each of stock solutions 2) 4) were added to about 800 mL purified water and madeup to 1000 mL with purified water. The pH of the final test water was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution.PREPARATION OF TEST FLASKSOne day before test start (Day -1), between 2400 and 2910 mL of untreated test medium was filled into 5 liter flasks (amber glass). To each flask, 90 mL activated sludge inoculum was added.The test media were aerated overnight with CO2-free air to purge the system of carbon dioxide.On the following day (Day 0), defined amounts of the test item were directly added to the test flasks. No emulsifiers or solvents were used.The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 770 mg sodium benzoate per 100 mL test water (purged with CO2-free air) was prepared. From this, 10 mL aliquots were added to the corresponding test flasks.The test flasks were made up to a volume of three liters with test water (purged with CO2-free air). Two absorber flasks, the first one containing 300 mL 0.05 M NaOH and the second one containing 200 mL 0.05 M NaOH, were connected in series to the exit air line of each test flask.TEST CONDITIONSTest vessels:5-liter all-glass amber bottles.Identification:The test vessels were labeled with the necessary information to ensure unmistakable identification.Test duration:28 daysLight conditions:The test vessels were incubated in a dark room.Test temperature:21 - 22 °C. The inoculated flasks were incubated in a temperature-controlled room. The temperature was checked on each sampling date (see Section 3.6.1) in a separate flask with purified water. Additionally, the room temperature was continuously recorded.CO2-free air:Air was led through a bottle containing about 750 mL of a 2 M NaOH solution to trap CO2. The CO2-free air was passed through the test solutions at a rate corresponding to about 30–100 mL/min.pH:Prior to test start (Day 0), the pH was measured in each test flask after the addition of test and/or reference item and, if necessary, adjusted to 7.4 ± 0.2. At the end of incubation (Exposure Day 28), the pH was measured again in each test flask.DETERMINATION OF CO2Sampling dates:Test item and inoculum control:Exposure Day 2, 5, 7, 9, 12, 14, 19, 23, 27, 28 and 29 Procedure control:Exposure Day 2, 7, 14, 28 and 291Toxicity control:Exposure Day 7, 14, 28 and 291On each sampling day, an aliquot of 5.0 mL was withdrawn from the absorber flask nearest to the test flask for analysis of inorganic carbon (IC). Additional samples for analysis of IC were withdrawn from the second absorber flask of all test vessels on Exposure Day 14 and at the end of the exposure period on Exposure Day 28 in order to correct for any carry over of CO2 (see Section 3.6.3).After sampling on Exposure Day 28, the pH was measured in each test flask. Next, 1 mL concentrated HCl was added to each test flask and the flasks were aerated overnight with CO2-free air to drive off any residual CO2 present. On Day 29, a sample from each absorber flask was withdrawn and analyzed for IC to determine residual CO2 that was present in the test suspensions on Exposure Day 28. In this way, any residual CO2 remaining in the test suspensions was determined as the difference between the amounts of IC found before and after acidification.

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

Preliminary study:

Prior to test start, the solubility of the test item in test water was checked in a pre-test without GLP, and the test item was soluble at a concentration oabout 50 mg/L in test water. Therefore, IC and TC (inorganic and total carbon) were measured in the test item and inoculum flasks at the start of the test.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

77

Sampling time:

28 d

Details on results:

TEST ITEM:The percent biodegradation of the test item was calculated based on a total organic carbon content (TOC) of 0.592 mg C/mg test item. The CO2 formation of R0037857A in the test media significantly increased from the start of the test until test termination after 28 days. At the end of the 28 day exposure period, average biodegradation was 77%. Moreover, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was reached. In conclusion, R0037857A was found to be readily biodegradable under the test conditions within 28 days.TOXICITY CONTROL:The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the total organic carbon content (TOC) of the test item and the reference item. CO2 formation in the toxicity control showed a similar course over the 28 day exposure period as the two procedure controls, containing only the reference item. Within 14 days of exposure, biodegradation reached 72%. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 26 mg/L because biodegradation in the toxicity control was ≥25% within 14 days of incubation.DRIVING OFF CO2:No significant difference was found between the absolute amounts of IC measured on Exposure Day 28 and the absolute amounts of IC measured after acidification on Day 29. Consequently, no residual CO2 was present in the test solutions at the end of the test. pH MEASUREMENT AT THE END OF THE TEST: The pH measured in all flasks at the end of exposure (Day 28) was between 7.3 and 7.5.

BOD5 / COD results

Results with reference substance:

The percent biodegradation of the reference item was calculated based on a total organic carbon content (TOC) of 0.58 mg C/mg sodium benzoate.In the procedure controls, average biodegradation of the reference item was 82% by Exposure Day 14, thus confirming suitability of the activatedsludge (≥60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 87%.

Any other information on results incl. tables

	% Degradation
Time (days)	Test item			Reference item			Toxicity control
	Replicate No.			Replicate No.			Replicate No.
	1	2	Mean	1	2	Mean	1
2	3.4	5.0	4.2	38.0	35.7	36.8	--
5	10.6	12.2	11.4	--	--	--	--
7	14.6	17.5	16.0	68.5	68.6	68.5	47.6
9	24.5	23.4	24.0	--	--	--	--
12	59.2	36.5	47.9	--	--	--	--
14	63.6	61.0	62.3	82.5	81.5	82.0	72.3
19	71.7	72.8	72.3	--	--	--	--
23	75.7	75.3	75.5	--	--	--	--
27	76.3	78.5	77.4	--	--	--	--
28	75.7	77.4	76.5	89.6	83.7	86.7	84.5

 

--: No samples taken

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In a 28 Day CO2 Evolution (Modified Sturm) Test, the test item R0037857A was found to be readily biodegradable according to EU Commission Directive 92/69/EEC C.4-C, Commission Regulation (EC) No 440/2008, C.4 C and OECD Guideline for Testing of Chemicals, No. 301 B (1992).

Executive summary:

R0037857A was investigated for its ready biodegradability in a 28‑Day CO₂Evolution (Modified Sturm) Test according to EU Commission Directive 92/69/C.4-C, Commission Regulation (EC) No 440/2008, C.4‑Cand OECD Guideline for Testing of Chemicals, No. 301 B (1992).

 

R0037857A was found to be biodegradable by 77% under the test conditions within the 28‑day exposure period. Moreover, the pass level for ready biodegradability, i.e. a CO₂formation of at least 60% of the TOC in a 10‑day window within the 28‑day period of the test was reached.

 

In the toxicity control, containing both R0037857A and the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, R0037857A had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 26 mg/L.

 

In the procedure controls, average biodegradation of the reference item was 82% by Exposure Day 14, thus confirming suitability of the activated sludge (≥60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 87%.

 

 

Conclusion

In a 28‑Day CO₂Evolution (Modified Sturm) Test, the test item R0037857Awas found to be readily biodegradable according to EU Commission Directive 92/69/C.4-C,Commission Regulation (EC) No 440/2008, C.4‑Cand OECD Guideline for Testing of Chemicals, No. 301 B (1992).