Ethyl ethyl(phenyl)carbamate

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:

November 8, 2016 - December 6, 2016

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)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge:A sample of activated sludge was taken from the aeration tank of Sewage Treatment Plant”Czajka”, Warsaw receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was washed in the mineral medium. The coarse particles were removed by settling and the supernatant was discarded.
- Pretreatment: 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 22 °C, until application.
- Concentration of sludge: 30mg/L
- Water filtered: no

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
Stock solutions:
Prepare the following stock solutions, using analytical grade reagents.
(a) Monopotassium dihydrogen orthophosphate, KH2PO4 8.50 g
Dipotassium monohydrogen orthophosphate, K2HPO4 21.75 g
Disodium monohydrogen orthophosphate dihydrate Na2HPO4 · 2 H2O 33.40 g
Ammonium chloride, NH4Cl 0.50 g
Dissolve in water and make up to 1 litre The pH of the solution should be 7.4.
(b) Calcium chloride, anhydrous, CaCl2 27.50 g
Dissolve in water and make up to 1 litre
(c) Magnesium sulphate heptahydrate, MgSO4 · 7 H2O 22.50 g
Dissolve in water and make up to 1 litre.
(d) Iron(III) chloride hexahydrate, FeC13 · 6H2O 0.25 g
Dissolve in water and make up to 1 litre.
Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of solutions (b) to (d) and
make up to 11 with dilution water
- Test temperature: 22 ± 2°C
- pH: 7.27
- pH adjusted: NO
- Aeration of dilution water: yes
- Initial concentration of test item in medium : 100 mg/L
- Amount of suspended inoculum in 1 l of mineral solution:30mg/L


TEST SYSTEM
- Number of culture flasks/concentration:
Flasks# 21 and 22: Test suspension containing test item (100 mg/L) and inoculum 30 mg/L SS.
Flasks# 1, 2 and 3: Inoculum blank containing only inoculum 30 mg/L SS.
Flask# 4, 5 and 6: Procedure control containing reference item (sodium acetate 100 mg/L) and inoculum 30 mg/SS.
Flask# 23, and 24: Toxicity control: containing test item, reference item at the same concentrations as in the individual solutions and inoculum 30 mg/L SS.
Volume of test solution in flask,V: 0.164l
- Method used to create aerobic conditions: STIRRING
- Measuring equipment: closed WTW OxiTop OC 110 respirometer for BOD determination;
spectrophotometer Hach DR 3900 and 45600 reactor COD nitrate and nitrite concentration measurements;
thermo-cabinet WTW TS 606 CZ-G/3-VAR;
electronic temperature recorder EBI 310 –T ebro logger from Ebro GmbH;
pH-meter – multifunctional microcomputer meter ELMETRON CX-505;
computer to data transfer and for calculations;
- Test performed in closed system: yes
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used:
The CO2, evolved during test item degradation, was absorbed in a solution of potassium hydroxide. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of calculated ThOD of the test item.

SAMPLING
- Sampling frequency: 112 min during 28 days
- Sampling method: Readings taken at regular and frequent intervals.The consumption of oxygen was determined from the change in pressure in the apparatus.The data were recorded and stored in the measuring heads of the sample bottles.These collected data, using the controller, were infrared read out from the heads and stored in the controller. Using software Achat OC version 2.03 of Aqualytic the data were transferred from to controller to a computer .

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, run in parallel without test item . 3 replicates
- Toxicity control: yes, run in parallel 2 replicates
- Procedure control :3 replicates

Reference substance:

acetic acid, sodium salt

Remarks:

CAS/127-09-3, pure p.a.a min 98.5.White powder

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 17.7

Sampling time:

28 d

Details on results:

At the 28th day of the test the aerobic biodegradation of the testing item, the test item attained 17.7% of biodegradation. In a toxicity test the biodegradation was 38.0%. The oxygen uptake of the inoculum blank was equal to 36.9 mg/L and oxygen uptake of the test item was 47.7 mg/L (corrected because of nitrification) in 28 days and the pH values of all flasks were inside the range 7.19-8.33 . The test item can not be assumed to be inhibitory, because in the toxicity test, containing both of test item and reference item more than 25% of biodegradation (based on ThODHN4) occured in 28 days.

Results with reference substance:

The reference item reached 96.1% of biodegradation. Reference item has reached a pass level (60%) on day 5.

The mineral medium solution pH was equal to 7.06.

The preconditioned suspension of inoculum pH was equal to 7.27.

The initial concentration of test item in test flasks was equal to 100 mg/L.

Table No.2:Correction for oxygen uptake for interference by nitrification

days	0		28		difference
1) Concentration of nitrate (mg N-NO3/l)	#21	#22	#21	#22	#21	#22
	0.253	0.262	0.257	0.264	0.004	0.002
					0.003± 0.001
2) Oxygen equivalent (4.57× N-NO3) (mg/L)					0.014
3) concentration of nitrite  (mg N-NO2/l)	#21	#22	#21	#22	#21	#22
	0.015	0.016	0.013	0.013	0.001	0.000
					0.0005
4) Oxygen equivalent (3.43×N-NO2) (mg/L)					0.002
5) total oxygen equivalent 2) + 4)					0.016

Table No.3: pH values of test flask

flask #	21	22	1		2	3	4	5	6		23	24
	Test item			Control			Reference item			Toxicity test
initial	7.58	7.64	7.44		7.44	7.45	7.43	7.45	7.44		7.60	7.54
final	7.34	7.48	7.21		7.19	7.25	8.22	8.11	8.33		7.96	8.07

Table No.4 : Toxicity test

days	0		28		difference
1) Concentration of nitrate (mg N-NO3/l)	#23	#24	#23	#24	#23		#24
	0.246	0.248	0.251	0.254	0.002		0.003
					0.003
2) Oxygen equivalent (4.57× N-NO3) (mg/L)					0.023
3) concentration of nitrite  (mg N-NO2/l)	#23	#24	#23	#24		#23	#24
	0.015	0.015	0.014	0.013		0.000	-0.001
						-0.0005
4) Oxygen equivalent (3.43×N-NO2) (mg/L)					0.002
5) total oxygen equivalent 2) + 4)					0.023

Table No.5: 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	0.7	6.0	10.1	17.3	22.8	35.6	39.7	45.7	48.3	53.5	57.6	60.6	65.4
	a2	1.1	5.4	12.6	20.9	28.3	42.9	50.3	56.7	62.6	69.5	73.7	78.4	84.6
	am. avg	0.9	5.7	11.4	19.1	25.6	39.3	45.0	51.2	55.4	61.5	65.7	69.5	75.0
Blank test O2uptake. mg/L	b1	4.8	14.4	17.4	21.0	22.3	27.3	29.3	30.1	34.3	37.0	38.2	39.8	44.1
	b2	3.6	8.7	14.7	16.4	22.2	22.9	24.7	26.2	25.9	25.3	29.2	29.1	31.1
	b3	3.6	14.0	18.0	21.7	25.0	27.2	28.3	28.0	33.2	32.5	32.4	34.1	35.5
	bmavg	4.0	12.4	16.7	19.7	23.1	25.8	27.5	28.1	31.2	31.6	33.3	34.4	36.9
Reference item O2uptake. mg/L	w1	19.1	53.8	67.1	76.5	84.5	92.3	95.7	98.6	101.2	106.2	108.0	110.8	116.2
	w2	21.7	52.2	62.4	73.1	79.9	87.3	92.6	96.5	98.9	104.5	106.2	109.7	113.6
	w3	20.9	50.7	63.1	70.5	78.4	85.2	89.3	92.8	94.4	99.2	103.3	105.8	110.1
	wm. avg	20.5	52.2	64.2	73.3	80.9	88.3	92.5	95.9	98.2	103.3	105.8	108.7	113.3
Toxicity control O2uptake mg/L	a4tox1	1.8	62.9	74.9	83.8	99.3	115.9	127.9	140.7	149.6	163.5	171.6	177.2	186.5
	a5tox2	0.8	59.8	69.2	74.0	76.5	82.2	88.0	93.5	95.7	100.3	103.4	105.2	110.0
	toxm. avg	1.3	61.4	72.1	78.9	87.9	99.0	107.9	117.1	122.7	131.9	137.5	141.2	148.3
Corrected test item O2uptake, mg/L	a1- bm	-3.3	-6.4	-6.6	-2.4	-0.4	9.8	12.2	17.6	17.1	21.9	24.3	26.3	28.5
	a2- bm	-2.9	-7.0	-4.1	1.2	5.2	17.1	22.8	28.6	31.4	37.9	40.5	44.0	47.7
Reference item % degradation ThOD = 0.78 mgO2/mg C = 100 mg/L	R1(w1)	19.4	53.1	64.6	72.8	78.7	85.3	87.5	90.3	89.9	95.6	95.8	98.0	101.6
	R2(w2)	22.6	51.1	58.7	68.4	72.8	78.9	83.5	87.6	86.8	93.4	93.5	96.6	98.4
	R3(w3)	21.6	49.2	59.5	65.1	70.8	76.1	79.3	82.9	81.1	86.7	89.7	91.6	93.8
	Rtoxavg	22.1	50.1	59.1	66.7	71.8	77.5	81.4	85.2	84.0	90.0	91.6	94.1	96.1

The reference item biodegradability result R1(w1 ) is not taken into average value calculations, as it is greater than 100% on 28th day.

Calculations:

This yields the BOD expressed as mg oxygen/mg test item:

BOD =mg O₂uptake by test item - mg O₂uptake by blank sample / mg test item in flask = mg O₂per mg test item

The biodegradation level (%) can be calculated from:

% biodegradation % ThOD = (BOD (mg O₂/mg test item) / ThOD (mg O₂/mg test item) )*100

Test item is a nitrogen-containing substance and maz affect the oxygen uptake because of nitrification.

In the case of complete nitrification (transformation of ammonium to nitrate), the following

equation applies:

NH4⁺+ 2O₂→ NO3^-+ 2H⁺+ H₂O

The oxygen taken up by 14 g of nitrogen is 64 g and thus the oxygen consumed in nitrate

formation is 4.57 × increase of nitrate-N concentration.

If incomplete nitrification takes place, the following equations apply:

NH4⁺+ 3/2 O₂→ NO₂^-+ 2H⁺+ H₂O

NO₂^-+ 1/2 O₂→ NO₃^-

In this case the observed oxygen uptake by the reaction mixture may be corrected for theamount of oxygen used in oxidizing ammonium to nitrite and nitrate.

O2 consumed in nitrate formation=4.57* increase in nitrate concentration

O2 consumed in nitrite formation=3.43* increase in nitrite concentration

O2 lost in nitrite disappearance=-3.43* decrease in nitrate concentration

So that, O2 uptake due to nitrification0 +-3.43*change in nitrite concentration+4.57* increase in nitrate concentration and thus

O2uptake due to C oxidation=total observed uptake-uptake due to nitrification. The above corrected value for oxygen consumption due to C oxidation can be compared withThOD_Nh4whereareas the uncorrected value for oxygen consumption can be compared withThOD_N03.

The test item can be expressed as CcHhClclNnNanaOoPpSs

ThOD_N03=[16(2C+0.5(H-Cl)+2.5N+3S+2.5P+0.5NA-O) / test item molecular weight ]mgO₂/mg test item

ThOD_Nh4=[16(2C+0.5(H-Cl-3N)+3S+2.5P+0.5NA-O) / test item molecular weight ]mgO₂/mg test item

The corrected (for nitrification and for that by the blank inoculum control after the same time) BOD values for oxygen consumption due to C oxidation could then be compared with

ThOD NH4, whereas the total observed O2 uptake (BOD corrected only for that by the blank inoculum control after the same time) can be compared with ThODNO3 (complete nitrificationassumed). Because of the known test item composition the ThOD could be calculated and to biodegradation calculation ThODNO3 was used.

The calculated ThOD_NH4= 2.15 mgO₂/mg test item and ThOD_NO3= 2.48 mgO₂/mg test item.

During the performed test the values of BOD directly obtained, in apparatus, are expressed in mg O₂/l of test solution. This way the biodegradation level (%) can be calculated using this equation:

mg/l O₂uptake by test item-mg/l O₂upatek by blank sample –uptake due to nitrification mg/lO₂/ThOD_Nh4(mgO₂/mg test item)* concentration of the test item (mg test item/l).

After correction for oxygen uptake for nitrification and for the blank inoculum control, the following biodegradation and toxicity calculation is suggested on the 28th day:

TEST ITEM BIODEGRADABILITY:

((75.0-36.9-0.016) mgO₂/l / ThOD_Nh4*conc.of test item )*100%=[5.24 mgO₂/l / (2.15 mgO₂/mg test item* 100.0 mg test item/l)]*100%= 17.7%

TOXICITY TEST BIODEGRADABILITY:

[(148.3 - 36.9 - 0.023) mgO₂/l / 100.0 mg test item/l*2.15 mgO₂/mg test item+0.78 mgO₂/ mg sodium acet .*100mgsodium acet . /l ]*100% =[111.38 mgO₂/l / 215mgO₂/ l 78.0mg O₂/ l ]*100%=38%

The nitrification effects on the 28th day are very modest (see table No.2) and it was assumed that those effects on the 14th day are the same as the final ones. Probably, the test item is an inhibitor of nitrification process.

Validity criteria fulfilled:

yes

Remarks:

Reference item has reached the pass level (60%) on day 5. The oxygen uptake of the inoculum blank is 36.9 mg O2/l in 28 days.4 The pH were inside the range 6-8.5 and the oxygen consumption by the test item is not > 60%.replicate difference is < 20%.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

At the 28th day of the test the measured aerobic biodegradation of the test item equals 17.7%. Thus, the test item cannot be deemed to be readily biodegradable.

Executive summary:

In accordance with OECD TG 301F and GLP study, the test of aerobic biodegradability was determined using manometric respirometry. Mineral solution containing 100 mg/L of test item was inoculated with microorganisms and incubated under aerobic conditions (oxygen presence) in a closed respirometer flask at constant temperature of 22 ± 2°C for 28 days. The blank tests were run in parallel with only inoculum. A reference item was run in parallel to check the operation of the procedures and toxicity test was run in parallel to check possible inhibitory effect. The degradation was followed by the determination of oxygen uptake (read out every 112 min) which was expressed as a percentage of calculated ThOD of the test item. 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. In a toxicity test, containing both the test item and a reference item, on the 28th day the biodegradation was 38%. Thus the test item can be assumed to be inhibiotry.The recorded measurement temperature didn’t exceed by 0.40 °C the expected 22 °C. According to OECD criteria, the pass level for ready biodegradability is 60 %, test item attained only 17.7 % of biodegradation and is no readily biodegradable.

Description of key information

Key study: OECD TG 301F and GLP study. At the 28th day of the test the measured aerobic biodegradation of the test item equals 17.7%. Thus, the test item cannot be deemed to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Key study: In accordance with OECD TG 301F and GLP study the test of aerobic biodegradability was determined using manometric respirometry. Mineral solution of test item was inoculated with microorganisms and incubated under aerobic conditions at constant temperature of 22 ± 2°C for 28 days. The blank tests were run in parallel with only inoculum. A reference item and toxicity test were run in parallel. The degradation was followed by the determination of oxygen uptake which was expressed as a percentage of calculated ThOD of the test item. According to OECD criteria, the pass level for ready biodegradability is 60 %. Test item attained only 17.7 % of biodegradation and is no readily biodegradable.