Cytidine 3'-(dihydrogen phosphate)

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 17, 2017 - April 14, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A sample of activated sludge was taken from the aeration tank of Sewage Treatment Plant "Czajka", Warsaw, receiving predominantly domestic sewage.
- Preparation of inoculum: The coarse particles were removed by settling and the supernatant was discarded. The sludge was washed in the mineral medium. The concentrated sludge was suspended in mineral medium to yield a concentration of 3-5 g suspended solids/l and it was aerated for 5 days, at the test temperature of 22ºC, until application. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: 10ml of solution (A) in 800 ml water, plus 1 ml solutions (B), (C), (D) and 11ml water. The following stock solutions were used, prepared with analytical grade reagents: Solution (A) contains: 8.50 g monopotassium dihydrogen orthophosphate (KH2PO4), 21.75 g dipotassium monohydrogen orthophosphate (K2HPO4), 33.40 g disodium monohydrogen orthophosphate dihydrate (Na2HPO4·2H2O), 0.50 g ammonium chloride (NH4Cl), in 1 L double-distilled water. Solution (B) contains: 27.50 g calcium chloride, anhydrous (CaCl2) in 1L water. Solution (C) contains: 22.50 g magnesium sulphate heptahydrate (MgSO4·7H2O) in 1L water. Solution (D) contains: 0.25 g iron(III) chloride hexahydrate (FeCl3·6H2O) in 1L water.
- The water used is double-distilled, containing about 3 mg/L of organic carbon (< 10% of the organic carbon content introduced by the test item), checked by DOC analysis using spectrophotometer Hach DR 3900 and Hach-Lange reagents.
- Test temperature: 22 ± 2ºC
- pH: 7.4 ± 0.2
- pH adjusted: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes
- Volume of test solution in flask, V:0.164 L

TEST SYSTEM
- Number of culture flasks/concentration: triplicates were used, flasks #7,8,9 containing test item (100 mg/l) and inoculum (30 mg/l SS)
- Measuring equipment: O2 uptake was measured by a closed WTW OxiTop OC 110 respirometer
- Details of trap for CO2 and volatile organics if used: potassium hydroxide solution.

SAMPLING
- Sampling frequency: The data were read out every 112 min during the 28 day test (40 320 min that is 360 readings) and were recorded and stored in the measuring heads of the sample bottles.

CONTROL AND BLANK SYSTEM
- Inoculum blank: flasks #1,2,3, containing only inoculum 30 mg/L SS
- Procedure control: flasks #4,5,6, containing the reference item (sodium acetate 100 mg/L) and inoculum 30 mg/SS.
- Toxicity control: flasks #10,11,12, containing test item, reference item and inoculum 30 mg/L SS.

STATISTICAL METHODS: The calculations and the graphs were performed using SigmaPlot 9.0 software of SYSTAT Software, Inc., USA purchased from GAMBIT CoiS Ltd, Poland.

Reference substance:

acetic acid, sodium salt

Remarks:

CAS 127-09-3, purity 99.7, CHEMPUR

Test performance:

In this test item as a nitrogen-containing substance may affect the oxygen uptake because of nitrification reaction. The observed oxygen uptake by the reaction mixture may be corrected for the amount of oxygen used in oxidizing ammonium to nitrite and nitrate.

Key result

Parameter:

% degradation (O2 consumption)

Value:

99.1

Sampling time:

28 d

Details on results:

- At the 28th day of the test the aerobic biodegradation of the testing, the test item attained 99.1% of biodegradation. The pH values of all flasks were inside the range 6.68-8.58.
- In a toxicity test, containing both the test item and a reference item more than 25 of biodegradation (based on ThODNH4) occurred in 14 days. Therefore, the test item cannot be assumed to be inhibitory.

Results with reference substance:

The reference item reached 72.4% of biodegradation and the level for ready biodegradability by 5 days.

Test item

Table 2.1 Correction for oxygen uptake for interference by nitrification

days	0	28	difference
1) Concentration of nitrate (mg N-NO3/l)	Flask #9	Flask #9	Flask #9
	1.78	15.6	13.82
2) Oxigen equivalent (4.57 x N-NO3) (mg/L)			63.16
3) Concentration of nitrite (mg N-NO2/l)	Flask #9	Flask #9	Flask #9
	0.016	0.077	0.061
4) Oxigen equivalent (3.43 x N-NO2) (mg/L)			0.209
5) total oxigen equivalent 2) + 4)			63.37

Samples no. 7 and 8 were not analysed because of an incident.

Toxicity test

Table 2.2. Correction for oxygen uptake for interference by nitrification

days	0	28	difference
1) Concentration of nitrate (mg N-NO3/l)	Flask #11	Flask #11
	0.22	12.10	11.88
2) Oxigen equivalent (4.57 x N-NO3) (mg/L)			54.29
3) Concentration of nitrite (mg N-NO2/l)	Flask #11	Flask #11
	0.018	0.041	0.023
4) Oxigen equivalent (3.43 x N-NO2) (mg/L)			0.079
5) total oxigen equivalent 2) + 4)			54.37

Samples no. 10 and 12 were not analysed because of an incident.

Table 3. The pH values of test flasks

Flask #	7	8	9	1	2	3	4	5	6	10	11	12
	Test item			Inoculum blank			Reference item			Toxicity test
Initial	7.20	7.21	7.21	7.59	7.54	7.65	7.61	7.64	7.62	7.12	7.21	7.21
final	6.96	7.00	6.68	7.20	7.19	7.20	8.48	8.58	8.28	7.14	*	7.61

* missed measurement by mistake. No adjustment of pH was conducted

Table 4. Sample oxygen uptake: biodegradability

		Time, days
		1	3	5	7	9	12	14	16	18	21	23	25	28
Test item O2uptake, mg/L	a1	1.7	41.6	96.2	130.5	143.8	158.8	163.8	169.1	173.4	182.3	186.6	190.9	197.0
	a2	0.8	30.5	74.6	117.3	140.2	152.5	158.6	163.8	168.9	174.2	180.8	183.9	189.6
	a3	0.0	29.9	77.4	126.6	138.0	151.1	154.8	158.6	161.6	169.4	174.2	177.8	182.2
	amavg	0.9	34.0	82.7	124.8	140.7	154.2	159.1	163.8	168.0	175.3	180.5	184.2	189.6
Blank test O2uptake, mg/L	b1	4.9	10.7	19.6	27.5	31.8	38.0	38.8	43.5	46.5	50.3	52.4	55.0	57.9
	b2	4.0	12.0	18.8	22.7	26.2	32.3	34.0	37.5	40.7	46.2	48.9	52.1	56.3
	b3	6.9	14.9	19.5	23.2	27.1	29.3	29.9	31.4	32.5	38.2	38.6	41.1	44.4
	bmavg	5.3	12.5	19.3	24.5	28.4	33.2	34.2	37.4	39.9	44.9	46.6	49.4	52.9
Reference item O2uptake, mg/L	w1	13.7	50.4	64.5	72.9	77.3	84.5	86.7	88.1	90.8	94.6	96.6	97.2	98.7
	w2	15.0	52.8	67.3	76.5	82.1	89.8	93.1	95.7	98.7	103.0	105.8	109.0	111.4
	w3	13.2	52.4	67.6	79.0	83.3	90.0	93.1	98.0	100.3	106.7	110.3	113.1	118.0
	wmavg	14.0	51.9	66.5	76.1	80.9	88.1	91.0	93.9	96.6	101.4	104.2	106.4	109.3
Toxicity control O2uptake, mg/L	a4tox1	10.3	82.5	139.2	175.8	189.8	202.9	205.0	208.6	213.1	219.5	222.9	225.3	230.2
	a5tox2	13.0	80.2	119.4	161.8	190.1	205.4	211.4	217.6	223.0	234.3	238.2	242.8	250.0
	a5tox3	9.0	74.3	120.5	167.6	194.9	207.5	217.4	Nitrification measurement on 14^thday
	toxmavg	10.8	79.0	126.4	168.4	191.6	205.3	211.3	213.1	218.0	226.9	230.6	234.0	240.1
Corrected test item O2uptake, mg/L	a1- bm	-3.5	29.1	76.9	106.0	115.5	125.6	129.5	131.7	133.5	137.4	139.9	141.5	144.1
	a2- bm	-4.4	18.0	55.3	92.8	111.8	119.3	124.4	126.3	128.9	129.3	134.2	134.5	136.8
	a3- bm	-5.3	17.4	58.1	102.1	109.7	117.9	120.6	121.1	121.7	124.4	127.6	128.4	129.3
Reference item % biodegradation (BOD/ThODxC)x100   ThOD=0.78 mgO2/mg C=100 mg/L	R1(w1)	10.8	48.5	57.9	62.0	62.8	65.8	67.2	64.9	65.3	63.6	64.0	61.2	58.7
	R2(w2)	12.4	51.6	61.5	66.6	68.9	72.6	75.5	74.6	75.4	74.5	75.9	76.4	75.0
	R3(w3)	10.2	51.1	62.0	69.8	70.4	72.8	75.5	77.6	77.4	79.2	81.6	81.7	83.5
	Rtoxavg	11.2	50.4	60.5	66.2	67.4	70.4	72.8	72.4	72.7	72.4	73.9	73.1	72.4

 

Validity criteria fulfilled:

yes

Remarks:

Difference of replicates <20%,reference item reached pass level (60%) on day 5, biodegradation of toxicity test on day 28 was 95.3%,oxigen uptake of blank was 52.9 mg O2/l in 28 days, pH values were inside 6-8.5 range,oxigen consumptiom by test item <60%

Interpretation of results:

readily biodegradable

Conclusions:

At the 28th day of the test the measured aerobic biodegradation of the test item equals 99.1%. Therefore, the test item can be deemed to be readily biodegradable.

Executive summary:

The study of ready biodegradability of test substance in an aerobic aqueous medium with manometric respirometry method was performed according to OECD 301F and EU C.4 -D method, following GLP. 100 mg/l of test item was inoculated with activated sludge (30 mg/L SS) and incubated under aerobic conditions in a closed respirometer flask at constant temperature (22 ± 2ºC) for 28 days. The observed oxygen uptake by the reaction mixture was corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate. All the acceptability criteria were met and the study was considered valid. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. At the 28th day, the reference item reached 72.4% of biodegradation and the level for ready biodegradability by five days. In a toxicity test, containing both the test item and a reference item, on the 28th day the biodegradation was 72.5%. Thus, the test item is not inhibitory. At the 28th day of the test the measured aerobic biodegradation of the test item equals 99.1%. Therefore, the test item can be deemed to be readily biodegradable.

Description of key information

Key study: OECD 301F and EU method C.4 -D. GLP study. At the 28th day of the test the measured aerobic biodegradation of the test item equals 99.1%. Therefore, the test item can be deemed to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Key study: The study of ready biodegradability of test substance in an aerobic aqueous medium with manometric respirometry method was performed according to OECD 301F and EU C.4 -D method, following GLP. 100 mg/l of test item was inoculated with activated sludge (30 mg/L SS) and incubated under aerobic conditions in a closed respirometer flask at constant temperature (22 ± 2ºC) for 28 days. The observed oxygen uptake by the reaction mixture was corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate. All the acceptability criteria were met and the study was considered valid. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. At the 28th day, the reference item reached 72.4% of biodegradation and the level for ready biodegradability by five days. In a toxicity test, containing both the test item and a reference item, on the 28th day the biodegradation was 72.5%. Thus, the test item is not inhibiotry. At the 28th day of the test the measured aerobic biodegradation of the test item equals 99.1%. Therefore, the test item can be deemed to be readily biodegradable.