[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25/10/2017 - 22/11/2017

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

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: 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 sludge was aerated for 7 days, at the test temperature of about 22ºC, until application. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids. Before an application the sludge was washed in a mineral medium.
- Amount of inoculum suspended: 30 mg/L.

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.
- Test temperature: 22 ± 2°C
- pH: 7.3 ± 0.2
- pH adjusted: no
- Volume of test solution in flask, V: 0.164 L
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: sample bottles in closed WTW OxiTop OC110 respirometer.
- Number of culture flasks/concentration: triplicates were used, flasks #13, 14, 15 containing test item (100 mg/l) and inoculum 30mg/L SS.
- Measuring equipment: O2 uptake was measured by the closed WTW OxiTop OC110 respirometer. The data was read out every 112 min during the 28 day test (40 320 min that is 360 readings) and was recorded and stored in the measuring heads of the sample bottles.
- Test performed in closed vessels due to significant volatility of test substance: no.
- Details of trap for CO2 and volatile organics if used: Three flakes of potassium hydroxide were added to each of the CO2-absorber compartments.

SAMPLING
- Sampling frequency: The oxygen uptake was determined from the readings taken by the equipment at regular intervals (every 112 min).

CONTROL AND BLANK SYSTEM
- Inoculum blank: flasks #1, 2, 3, containing only inoculum 30mg/L SS.
- Procedure control: flasks #4, 5, 6, containing reference item (sodium acetate 100 mg/l) and inoculum 30mg/L SS.
- Toxicity control: flasks #16, 17, 18, containing test item, reference item and inoculum 30mg/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:

purity 99.7% (CHEMPUR)

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

- At the 28th day of the test the aerobic biodegradation of the testing, no biodegradation was observed. The pH values of all flasks were inside the range 7.02-8.42.
- In the toxicity test, containing both the test item and reference item, the biodegradation did not reach 25% (21.7% based on ThODNH4) in 14 days. Therefore, the test item might be inhibitory.

Results with reference substance:

The reference item reached 85.0% of biodegradation by day 4.

Table 2. Sample oxygen uptake: biodegradability.

		time, days
		1	3	5	7	9	12	14	16	18		21		23	25		28
Test item O2 uptake, mg/L	a1	8.8	14.5	16.9	20.7	22.2	24.6	24.8	25.7	26.7		28.7		29.8	30.9		33.4
	a2	5.9	11.4	14.4	16.9	19.9	22.1	23.6	25.3	27.4		29.9		31.0	32.7		34.0
	a3	2.7	8.1	11.6	15.3	16.9	20.7	21.7	24.6	26.8		30.2		31.5	33.6		36.8
	am. avg	5.8	11.4	14.3	17.6	19.7	22.5	23.4	25.2	26.9		29.6		30.8	32.4		34.7
Blank test O2 uptake, mg/L	b1	5.8	13.5	16.9	20.9	23.3	26.6	28.1	30.1	33.0		34.8		37.0	38.9		42.1
	b2	7.4	12.6	17.5	21.7	24.1	27.3	28.9	31.2	32.3		35.0		36.6	38.0		40.4
	b3	9.1	13.3	15.7	19.9	23.7	26.5	26.8	28.4	30.7		33.5		35.6	37.3		41.5
	bm avg	7.4	13.1	16.7	20.8	23.7	26.8	27.9	29.9	32.0		34.5		36.4	38.1		41.3
Reference item O2 uptake, mg/L	w1	24.5	57.6	73.2	83.8	90.5	95.8	98.7	101.3	105.3		110.1		113.4	113.8		117.8
	w2	17.8	53.4	70.2	79.2	84.1	88.0	91.4	92.8	95.7		98.5		100.0	101.2		104.0
	w3	17.5	52.8	66.4	76.4	80.5	86.9	88.4	90.7	92.8		96.6		97.1	98.4		101.2
	wm. avg	19.9	54.6	70.0	79.8	85.0	90.2	92.8	94.9	97.9		101.8		103.5	104.5		107.7
Toxicity control O2 uptake, mg/L	a4tox1	9.6	54.7	70.5	80.5	87.0	91.3	95.4	98.5	101.2		106.2		108.7	110.9		114.3
	a5tox2	10.0	52.3	67.5	77.7	82.4	87.2	90.2	93.1	93.3		96.8		98.6	98.5		101.3
	a6tox3	11.5	55.7	70.1	80.4	87.6	93.4	95.7	98.7		101.2	104.0	107.1		109.9	112.8
	toxm. avg	10.3	54.2	69.4	79.5	85.6	90.7	93.8	96.8	98.5		102.4		104.8	106.4		109.5
Corrected test item O2 uptake, mg/L	a1 - bm	2.1	41.5	53.8	59.6	63.3	64.5	67.4	68.7	69.2		71.8		72.3	72.8		73.0
	a2 - bm	2.5	39.2	50.8	56.8	58.7	60.4	62.2	63.2	61.3		62.4		62.2	60.4		60.0
	a3 - bm	4.0	42.5	53.4	59.6	63.9	66.6	67.8	68.8	69.2		69.5		70.7	71.8		71.5
Reference item % degradation - ThOD = 0.78 mgO2/mg; C = 100 mg/L	R1(w1)	21.8	56.9	72.5	80.7	85.7	88.5	90.8	91.6	94.0		97.0		98.7	97.1		98.0
	R2(w2)	13.2	51.6	68.7	74.8	77.5	78.5	81.3	80.7	81.7		82.1		81.5	80.9		80.3
	R3 (w3)	12.8	50.8	63.8	71.3	72.8	76.9	77.5	78.0	77.9		79.7		77.8	77.3		76.8
	Rtoxavg	16.0	53.1	68.3	75.6	78.7	81.3	83.2	83.4	84.5		86.3		86.0	85.1		85.0

Table 3. pH values of test flasks (no adjustment of pH was conducted).

flask #	13	14	15	1	2	3	4	5	6	16	17	18
	Test item			Inoculum blank			Reference item			Toxicity test
initial	7.60	7.60	7.60	7.58	7.58	7.59	7.58	7.58	7.58	7.59	7.58	7.58
final	7.10	7.15	7.15	7.02	7.10	7.13	8.26	8.35	8.42	8.01	8.21	8.14

Table 4. Correction for oxygen uptake for interference by nitrification.

days	0	14	difference
1) Concentration of nitrate (mg N-NO3/l)	Flask #16	Flask #16	Flask #16
	0.192	0.931	0.739
2) Oxygen equivalent (4.57× N-NO3) (mg/L)			3.377
3) concentration of nitrite (mg N-NO2/l)	Flask #16	Flask #16	Flask #16
	0.022	0.018	-0.004
4) Oxygen equivalent (3.43×N-NO2) (mg/L)			-0.014
5) total oxygen equivalent 2) + 4)			3.363

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks'.

Interpretation of results:

not readily biodegradable

Conclusions:

At the 28th day of the test, no biodegradation was observed. Thus, the test item is not readily biodegradable.

Executive summary:

A 28-day ready aerobic biodegradability test in an aerobic aqueous medium with manometric respirometry method was performed on the test item, according to OECD 301F / EC C.4 – D manometric respirometry methods, in accordance with GLP principles. 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. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. In the toxicity test, containing both the test item and a reference item, on the 14th test day the biodegradation (based on ThOD) attained 21.7%. Thus, the test item might be inhibitory. At the 28th day of the test the measured aerobic no biodegradation was observed (the test item attained 0%). Thus, the test item is not readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

nitrification not considered.

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): effluent of the municipal STP in Wyhl (Abwasserzweckverband Kaiserstuhl Nord, Wyhl, Germany; 14 300 inhabitant equivalents).
- Concentration of sludge: Two drops of inoculum were added to 1 L of medium.
- Initial cell/biomass concentration: about 500 colony forming units per mL (CFU/mL).

Duration of test (contact time):

28 d

Initial conc.:

3.27 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- The CBT was performed according to the conditions specified in the test guidelines.
- pH adjusted: no

TEST SYSTEM
- Number of culture flasks/concentration: 2
- Measuring equipment: oxygen electrode (Oxi 196 with EO 196-1.5, WTW Weilheim, Germany).
- Other: Two parallel tests were run for each of 4 series.

SAMPLING
- Sampling frequency: oxygen concentration in the test vessels was monitored at day 0, 0 (after 3 h), 1, 7, 14, 21 and 28. pH values were monitored during the course of the test.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes. The blank series contained only mineral medium and inoculum.
- Abiotic sterile control: no.
- Toxicity control: yes. In addition to the test compound, this series also contained sodium acetate.
- Other: The quality control series contained readily biodegradable sodium acetate as the only respective carbon sources beside the inoculum.

Reference substance:

acetic acid, sodium salt

Remarks:

ThOD of 5 mg/L

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

-In accordance with the test guidelines the test item was not readily biodegradable in the CBT (0%).
- Primary elimination monitored by HPLC–UV–VIS was 100% for the test item within 7 days.
- Toxicity of the test compounds against degrading bacteria was not detected; elimination by adsorption of the test compounds onto the bacterial mass or glass surface was not detected.

Results with reference substance:

The reference substance achieved ≥ 60% decomposition within 14 days.

Validity criteria fulfilled:

yes

Remarks:

all validity criteria were within the ranges specified in the guidelines.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under test conditions, no biodegradation of the test item was observed (0%). Therefore, the test item is not readily biodegradable.

Executive summary:

An aerobic ready biodegradability Closed Bottle Test was performed on the test item, according to OECD 301D (non-GLP). A solution of 3.27 mg/L test item was inoculated with a relatively small number of micro-organisms from a mixed population (effluent from municipal STP, about 500 CFU/mL) and kept in completely full, closed bottles in the dark at constant temperature for 28 days, and O2 consumption was monitored. A blank test, a quality control test with reference substance (sodium acetate) and a toxicity test were run in parallel. Primary elimination of the test item (100%) occurred within 7 days, showing abiotic degradation. No toxicity of the test item was observed in the controls, so the test item is not inhibitory. Under test conditions, no biodegradation of the test item was observed (0%). Therefore, the test item is not readily biodegradable.

Reference 3

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

yes

Remarks:

test lasted 30 days.

GLP compliance:

not specified

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): The sludge was obtained from a municipal STP (Abwasserzweckverband Breisgauer Bucht, Forchheim, Germany; 650000 inhabitant equivalents). The dry matter content was 4.7 g/L.
- Storage conditions: The sludge was stored in the laboratory at room temperature, aerated until use and washed three times with tap water to reduce the DOC background.
- Preparation of inoculum for exposure:
- Pretreatment:
- Concentration of sludge:
- Initial cell/biomass concentration:
- Water filtered: yes/no
- Type and size of filter used, if any:

Duration of test (contact time):

30 d

Initial conc.:

50 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Test temperature: 21–25 ºC
- pH: 6.5 ≤ pH ≤ 7.5
- pH adjusted: yes. The pH was adjusted with NaOH or HCl.
- Aeration of dilution water: yes.
- Suspended solids concentration: The final dry matter content of sludge in all the test vessels was 1 g/L.

TEST SYSTEM
- Culturing apparatus: 2 L-Erlenmeyer vessels.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: gentle stirring and aeration.
- Measuring equipment: total organic carbon (TOC) analyser (TOC 5000, Shimadzu GmbH, Duisburg, Germany).
- Test performed in closed vessels due to significant volatility of test substance: no.

SAMPLING
- Sampling frequency: daily.
- Sampling method: Prior to measurement the samples were centrifuged for 30 min at 4000 U/min (Universal 32R, Hettich, Germany) and filtered (cellulose nitrate, 0.45 μm, Rohr, Göttingen,Germany) in order to detach carbon particles. DOC samples were analysed directly after taking and preparing the daily samples.
- Sample storage before analysis: Parts of the centrifuged and filtrated samples were stored deep-frozen (-20 ºC) until LC–MS/MS-analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Only mineral salts and inoculum were present in the blank.
- Abiotic sterile control: The abiotic negative control contained no sludge but instead highly toxic copper sulphate in order to measure potential abiotic elimination.
- Toxicity control: no.

Reference substance:

ethylene glycol

Remarks:

DOC of 50 mg/L.

Key result

Parameter:

% degradation (DOC removal)

Value:

ca. 12

Sampling time:

30 d

Details on results:

- Primary elimination monitored by HPLC–UV–VIS was 100% for the test item within 14 days. The primary elimination of the test item was rapid. Elimination kinetics were the same in both the biologically active test vessel and the abiotic control indicating an abiotic process being responsible for this step.
- HPLC–UV–VIS monitoring of the primary elimination of piperacillin showed two transformation products throughout the test, more polar than the parent compound (hydrolysed). It was possible to identify hydrolysed piperacillin as main product and bis-hydrolysed piperacillin as minor product of abiotic transformation products. The benzene moiety and the side chain with two tertiary nitrogen atoms were retained in this molecule.
- Toxicity of the test compounds against degrading bacteria was not detected; elimination by adsorption of the test compounds onto the bacterial mass or glass surface was not detected.

Table. Elimination of piperacillin in the ZWT; test vessel: sum of biotic and abiotic elimination; abiotic control: elimination due to abiotic process only.

Compound	Concentration (mgC/L)	Total elimination (DOC) after 28 days		Primary elimination (HPLC-UV-VIS, LC-MS/MS)
		Biotical active vessel (%)	Control (%)	Biotical active vessel (%)	Control (%)
Piperacillin	50	̴ 12	̴ 16	98.9	97.0

 

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Under test conditions, only a 12% biodegradation of the test item was observed. Therefore, the test item is not inherently biodegradable.

Executive summary:

A Zahn-Wellens test for inherent biodegradability was performed on the test item, according to OECD 302B (non-GLP). A mixture containing the test item (50 mg/L of DOC), mineral nutrients and a relatively large amount of activated sludge in aqueous medium is agitated and aerated at 20-25°C in the dark for 30 days, and DOC removal was monitored. A blank test, a quality control test with reference substance (sodium acetate) and a toxicity test were run in parallel. Primary elimination of the test item occurred within 14 days, showing abiotic degradation, probably due to hydrolysis. No toxicity of the test item was observed in the controls, so the test item is not inhibitory. Under test conditions, only a 12% biodegradation of the test item was observed. Therefore, the test item is not inherently biodegradable.

Description of key information

Weight of evidence. Based on the available information, the test item is not biodegradable.

A ready biodegradability study performed according to OECD 301F (GLP study), reports the test item as not readily biodegradable. Two other tests performed on the sodium salt of the substance, a Closed Bottle test performed according to OECD 301D (non-GLP) and a Zahn Wellens test performed according to OECD 302B (non-GLP) report the test item as not biodegradable (0 and 12% degradation, respectively), but susceptible to abiotic degradation, probably by hydrolysis.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Weight of evidence. Based on the available information, the test item is not readily or inherently biodegradable.

- An OECD 301F test. A 28-day ready aerobic biodegradability test performed according to OECD 301F / EC C.4 – D (GLP study) was performed with the test item. 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. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. In the toxicity test, containing both the test item and a reference item, on the 14th test day the biodegradation (based on ThOD) attained 21.7%. Thus, the test item might be inhibitory. At the 28th day of the test the measured aerobic no biodegradation was observed (the test item attained 0%). Thus, the test item is not readily biodegradable.

- An aerobic ready biodegradability Closed Bottle Test was performed on the analogue substance piperacillin sodium, according to OECD 301D (non-GLP). A solution of 3.27 mg/L test item was inoculated with a relatively small number of micro-organisms from a mixed population (effluent from municipal STP, about 500 CFU/mL) and kept in completely full, closed bottles in the dark at constant temperature for 28 days, and O2 consumption was monitored. A blank test, a quality control test with reference substance (sodium acetate) and a toxicity test were run in parallel. Primary elimination of the test item (100%) occurred within 7 days, showing abiotic degradation. No toxicity of the test item was observed in the controls, so the test item is not inhibitory. Under test conditions, no biodegradation of the test item was observed (0%). Therefore, the test item is not readily biodegradable. Based on the available information for the read-across approach, the target substance is not readily biodegradable.

- A Zahn-Wellens test for inherent biodegradability was performed on the analogue substance piperacillin sodium, according to OECD 302B (non-GLP). A mixture containing the test item (50 mg/L of DOC), mineral nutrients and a relatively large amount of activated sludge in aqueous medium is agitated and aerated at 20-25°C in the dark for 30 days, and DOC removal was monitored. A blank test, a quality control test with reference substance (sodium acetate) and a toxicity test were run in parallel. Primary elimination of the test item (100%) occurred within 14 days, showing abiotic degradation, probably due to hydrolysis. No toxicity of the test item was observed in the controls, so the test item is not inhibitory. Under test conditions, only a 12% biodegradation of the test item was observed. Therefore, the test item is not inherently biodegradable. Based on the available information for the read-across approach, the target substance is not inherently biodegradable.