4-benzyloxy-2-nitro-beta-pyrrolidinostyrene

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:

24/04/2017 to 24/05/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

adopted 17/07/1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 30/05/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pyrrolidine, 1-[2-[2-nitro-4-(phenylmethoxy)phenyl]ethenyl]-

Cas Number:

99474-22-3

Molecular formula:

C19H20N2O3

IUPAC Name:

Pyrrolidine, 1-[2-[2-nitro-4-(phenylmethoxy)phenyl]ethenyl]-

Specific details on test material used for the study:

- Analytical purity: > 98.4%
- Lot/batch No.: 0159115
- Expiration date of the lot/batch: 21/02/2018

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf (Date of collection: 21. Apr. 2017, batch no: 20170421). The sludge was filtrated, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use. The dry matter was determined as 4040 mg suspended solids/L.

Duration of test (contact time):

ca. 28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

>= 28.4 - <= 28.7 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

>= 20 - <= 20.2 mg/L

Based on:

other: calculated organic carbon

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Reference substance

Reference substance:

aniline

Results and discussion

Preliminary study:

No preliminary study performed

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 31

St. dev.:

9.5

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

Degradation of the positive control was 62 % after 8 days

Any other information on results incl. tables

Findings

Tables

IC-Values

 

In the following tables, the IC values (given in mg/L) which were measured in the samples of the front scrubber flasks are stated.

Table8.1‑a     IC values in mg/L of apparatus blanks, blank controls, front scrubber

Day	Apparatus blank 1	Apparatus blank 2	Blank Control 1	Blank Control 2
0	2.93	2.27	2.63	3.07
2	10.89	4.86	9.68	9.72
4	16.09	7.17	17.93	23.70
8	20.22	10.23	27.96	42.07
10	22.90	13.03	34.88	47.95
14	25.16	16.10	42.42	55.69
18	27.29	18.86	54.17	67.15
23	29.61	21.92	58.27	70.65
29	34.66	29.06	77.36	87.25

Table8.1‑b     IC values in mg/L of positive control, test flasks, front scrubber

Day	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.77	2.84	3.27	3.75	2.48	3.08
2	7.95	14.16	23.01	16.11	5.36	22.34
4	75.39	85.41	46.61	28.04	7.42	139.98
8	206.34	248.17	90.34	47.75	9.80	284.27
10	255.66	293.06	109.03	74.04	11.40	319.68
14	314.69	322.28	142.07	102.32	14.19	362.23
18	331.21	330.27	168.05	123.28	18.01	381.27
23	343.82	339.16	194.85	143.31	20.57	402.33
29	364.63	353.45	223.90	163.29	27.30	434.96

 

In the following tables, the IC values which were measured in the samples of the back scrubber flasks are stated.

Table8.1‑c     IC values in mg/L of blank controls, apparatus blanks, back scrubber

Day	Apparatus blank 1	Apparatus blank 2	Blank Control 1	Blank Control 2
0	3.01	2.67	2.59	2.83
29	3.45	4.62	5.50	3.69

Table8.1‑d     IC values in mg/L of positive control, test flasks, back scrubber

Day	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.73	2.38	2.75	3.21	3.03	2.84
29	3.29	2.87	3.63	3.87	3.65	3.51

 

Net IC

 

For each flask, the net IC was calculated by subtracting the mean IC value of the apparatus blanks of the corresponding sampling date from the remaining IC values. Exception: Values of day 0 do not need to be corrected.

The net IC values are presented in the following table.

Table8.1‑e     Net IC-values in mg/L of front scrubber flasks

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.6	3.1	2.8	2.8	3.3	3.8	2.5	3.1
2	4.4	4.4	2.7	8.9	17.7	10.8	0.1	17.1
4	8.9	14.7	66.4	76.4	37.6	19.0	-1.6	131.0
8	15.3	29.4	193.7	235.5	77.7	35.1	-2.8	271.6
10	19.5	32.6	240.3	277.7	93.7	58.7	-4.0	304.3
14	24.4	37.7	296.7	304.3	124.0	84.3	-3.8	344.2
18	33.7	46.7	310.7	309.8	147.6	102.8	-2.5	360.8
23	35.1	47.5	320.7	316.0	171.7	120.1	-2.6	379.2
29	48.1	58.0	335.4	324.2	194.6	134.0	-2.0	405.7

Table8.1‑f      Net IC-values in mg/L of back scrubber flasks

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.6	2.8	2.7	2.4	2.8	3.2	3.0	2.8
29	4.3	2.5	2.1	1.7	2.4	2.7	2.5	2.3

 

Negative values occur, when the apparatus blank was higher than the respective treatment. As the measured values in these blanks as well as in the abiotic control are very low, measurement uncertainties lead to negative degradation values in the abiotic control.

 

pH

 

In the following table, the pH at the end of the test (before addition ofHCl) is given:

Table8.1‑g     pH in Test flasks on day 28

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
28	7.6	7.6	7.4	7.4	7.5	7.5	7.0	7.3

Equations

Emitted carbon in mg/L test solution in the respective vessel at timetwas calculated using the following equation:

 

emittC = [[(IC(t) – IC(0)] *VolNaOH(t)] /VolTestVessel

 

with:

emittC                 emitted carbon in mg/L test solution

IC(t)                    net inorganic carbon in mg/LNaOHin the respective vessel at time t

IC(0)                   net inorganic carbon in mg/LNaOHin the respective vessel at the start of the test

VolNaOH (t)        remaining volumeNaOHin L in the scrubber at time t
(Volume at t = 0 (here: 0.1 L) - ∑ (all sample volumes up to time t))

VolTestVessel      test vessel volume in L (here: 1.5)

 

For day 29, the IC content of both scrubber flasks was taken into account.

Calculation of emitted carbon is necessary for the assessment of validity. The value obtained with this equation is multiplied with 3.667 (44/12) in order to obtain emitted CO₂.

 

The percentage biodegradation in the test flasks was calculated according to the guidelines.

 

Calculation Results

Emitted Carbon in mg/L

 

In the following table, the calculated emitted carbon is presented.

Table8.3‑a     Emitted carbon in mg/L

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
2	0.12	0.09	-0.01	0.40	0.95	0.47	-0.16	0.92
4	0.41	0.76	4.15	4.80	2.24	1.00	-0.27	8.35
8	0.82	1.71	12.35	15.05	4.81	2.03	-0.34	17.37
10	1.08	1.89	15.20	17.59	5.79	3.52	-0.41	19.28
14	1.38	2.19	18.61	19.09	7.65	5.10	-0.40	21.60
18	1.95	2.73	19.30	19.24	9.04	6.21	-0.31	22.42
23	2.01	2.75	19.71	19.42	*9.43	7.22	-0.31	23.32
29	2.90	3.35	20.36	19.66	10.57	7.96	-0.31	24.66

* Due to a leakage in the absorber flask, a slight loss ofNaOHwas observed on day 23. Therefore, the measured carbon concentrations after that date were corrected by taking into account the remaining volume in the flask (volume at the end of the test = 81 mL).

 

Degradation Values

 

In the following table, the percentage biodegradation is presented:

Table8.3‑b     Degradation values in %

Day	Positive Control 1	Positive Control 2	Positive Control Mean	Test 1	Test 2	Test Mean	Abiotic Control	Toxicity Control
2	-0.6	1.5	0.5	4.3	1.8	3.0	-0.8	2.0
4	17.9	21.2	19.5	8.3	2.0	5.2	-1.3	19.3
8	55.6	69.1	62.3	17.8	3.8	10.8	-1.7	40.0
10	68.8	80.7	74.7	21.5	10.0	15.8	-2.0	44.1
14	84.4	86.7	85.5	29.4	16.4	22.9	-2.0	49.2
18	85.0	84.7	84.8	33.6	19.1	26.3	-1.5	49.8
23	86.8	85.4	86.1	35.3	23.9	29.6	-1.5	51.9
29	86.4	82.9	84.6	37.3	23.9	30.6	-1.5	53.4

 

Because the values of day 29 are the sum of the IC values in scrubber flasks A and B, an increase (IC values in flasks B of the test higher than in those of the control) or a decrease (IC values in flasks B of the test lower than in those of the control) of degradation can be observed.

As the measured IC values in the abiotic control are very low, measurement uncertainties lead to negative degradation values while in fact no degradation has taken place.

 

Results and Validity

Results for the Test Item

¨          The test item is considered as “not readily biodegradable“.

¨          The degree of biodegradation reached 31 % after 28 days.

¨          The 10-day-window began on day 8, at its end, 26 % degradation were reached, missing the pass level of 60 % given in the OECD guideline.

¨          Degradation missed 60% within 28 days, too. Therefore, the test item is considered as “not ultimately biodegradablewithin 28 days”, as well.

¨          Abiotic degradation was not observed.

Validity

All validity parameters and values are presented in the following table:

Table9.2‑a     Validity

Parameter	Criterion	Found	Assessment
IC content of test item solution in medium	£5% of TC	0 %	valid
CO2emitted by the controls	< 70 mg/L	11.5 mg/L	valid
Difference within replicates	£20%	13.4 %	valid
Degradation of positive control > 60%	≤ 14 days	8 days	valid
Degradation in the toxicity flask on day 14	> 25%	49.2 %	valid

Discussion

All validity criteria were met.

Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation was not observed. Both replicates of the test item showed good correspondence.

If degradation in the toxicity flask is below 25% after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 49.2 % after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 28.9 mg/L”.

Ready biodegradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10%. Therefore, the test can be considered as “not readily biodegradable”.

No observations were made which might cause doubts concerning the validity of the study outcome.

 

The result of the test can be considered valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The following data were determined for the test item:
• 10-day-window: day 8 – 18
• Degradation at the end of 10-day-window: 26 %
• Degradation at the end of the test: 31 %
• Pass level following guideline: 60% at the end of 10-day-window for pure substances respective 60 % at the end of the test for mixtures

Therefore, following OECD 301B and EU C.4-C, the test item is not readily biodegradable in the 10-d window and within 28 days.

Executive summary:

The test item was GLP-tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 28.4 mg test item /L) in test medium following OECD 301B and EU-Method C.4-C. Aniline was chosen as positive control. Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L). The test was left running for 28 days.

All validity criteria were met. Degradation of the positive control was 62 % after 8 days.

The following data were determined for the test item:

•       10-day-window: day 8 – 18

•       Degradation at the end of 10-day-window: 26 %

•       Degradation at the end of the test: 31 %

•       Pass level following guideline: 60% at the end of 10-day-window for pure substances respective 60 % at the end of the test for mixtures

Therefore, following OECD 301B and EU C.4-C, the test item is not readily biodegradable in the 10-d window and within 28 days.