IPEMA

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:

2021-07-27 to 2021-08-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 1981, Revised 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Municipal waste water treatment plant
- Storage conditions: Activated sludge was continuously aerated at room temperature and
under laboratory diffused light until test start.
- Storage duration: max. 1 day
- Preparation of inoculum for exposure: Right before inoculation, the activated sludge was mixed
thoroughly to ensure homogeneity. For dry matter determination, 3 aliquots of 250 mL each were filtered under reduced pressure and dried at 105 °C. The dry substance determined was 2.08 g/L, from which 3.00 g dry substance/L was prepared by removing the supernatant solution. Within one day, the sludge was sampled and its dry weight was determined. After concentrating the activated sludge, the test medium was inoculated and aerated with CO2-free air overnight.
- Concentration of sludge: 30 mg/L

Duration of test (contact time):

29 d

Initial conc.:

299.6 mg/L

Based on:

test mat.

Details on study design:

TEST CONDITIONS
- Composition of medium and pH: see 'Any other information on materials and methods incl. tables'
- Test temperature: 19.37 – 20.97 °C with a mean temperature of 20.26 °C
- pH adjusted: not reported
- Suspended solids concentration: 30 mg suspended solids/L

TEST SYSTEM
- Culturing apparatus: temperature monitored laboratory environment
- Number of culture flasks/concentration: Test item group (test item and inoculum): 3 replicates
- Method used to create aerobic conditions: aeration
- Measuring equipment: Titration
- Details of trap for CO2: At intervals, the first trap in each train was disconnected and removed for titration with hydrochloric acid (0.05 M) to determine the residual hydroxide concentration. The second and third traps were moved up one position and a new bottle containing a barium hydroxide Ba(OH)2 solution attached to the end of the train.
Titrations were performed after 2, 5, 7, 9, 15, 23, 28, and 29 days after treatment. On day 28, the test vessels were opened and the pH measured. Each mixture was then acidified by adding 1.00 mL 37 % hydrochloric acid (HCl). The vessels were then re-sealed and the aeration continued overnight to purge any residual dissolved CO2 and carbonates before titrating all the remaining trap bottles of day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank (inoculum): 2 replicates
- Toxicity control (test item, reference item and inoculum): 1 replicate
- Other: Reference control group (reference item and inoculum): 2 replicates

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

62

Sampling time:

28 d

Details on results:

Biological results:
The reference item attained a degradation of 74 % after 28 d of exposure while the the test item showed a degradation of 62 % in the same period. After purging the carbon dioxide by adding concentrated hydrochloric acid from day 28 to 29, the reference substance was degraded by 74 % and the test item by 66 % respectively.
Although biodegradation of the test item was > 60 % after 28 d, this occurred outside the 10-d window (i.e. later than 10 d after degradation first reached 10 %) in the test item replicates.
In order to account for potential toxicity of the test item a toxicity control was conducted. No toxic effects can be assumed if at least 50 % degradation based on the reference or > 25 % in total within 14 d (assuming the reference and test items are dosed at the same C equivalent concentrations). In the toxicity control, biodegradation amounted to 54 % within 9 d and 63 % within 15 d, which is within the test guideline threshold of > 25 % after 14 d based on the reference control. Furthermore, biodegradation of the toxicity control amounted to 67 % after 28 d, which is > 80 % after 28 d based on the reference control.
The degradation of the test item increased to 10 % after day 2 and accounted for 33 % at the end of the 10-d window and reached finally 62% after day 28 and 66 % after day 29 respectively. Thus, IPEMA failed to degrade quickly enough to satisfy the 10-d window and qualify for classification as readily biodegradable because of this effect in present study.

Results with reference substance:

Pass level of the reference control group:
Reference item had to reach the pass level (60 %) within 14 d. In this study the degradation exceeded 60 % at day 9 in both reference item replicates.

Validity criteria:
Replicate variability of the test item group:
The difference of extremes in replicate values, for the removal of the test item at the plateau, at the end of the test or at the end of the 10-d window, should be less than 20 %. The divergence was < 20 % between the test item replicates at day 28.

Pass level of the reference control group:
Reference item had to reach the pass level (60 %) within 14 d. In this study the degradation exceeded 60 % at day 9 in both reference item replicates.

Toxicity control group:
If in the toxicity control, containing both the test item and a reference item, degradation is less than 25 % within 14 d, the test item can be assumed to be inhibitory. In the toxicity control which contained test item and reference item, biodegradation amounted from 54 % at day 9 to 63 % at day 14. This is higher than the 25 % trigger set by the test guideline.

Inoculum control:
CO₂ consumption in inoculum control should not exceed 70 mg/L after 28 d. In this study the mean carbon dioxide production recorded in the control vessels amounted to 55.2 mg/L after 28 d.

Pass Levels and Overall Degradation

Group	< 20 % difference in replicates	Reference control > 60 % by day 14	CO2 in Inoculum control < 70 mg/L by day 28	pH range within 6.0-8.5	Maximum mean degradation At day 28 (29)
Inoculum control group	Yes	-	Yes	Yes	-
Reference control group	Yes	Yes (already at day 9)	-	Yes	74 (74)
Test item group	Yes	-	-	Yes	62 (66)
Toxic control group	-	-	-	Yes	67 (68)

-     Not applicable

pH-Values

Group	Day 28
Inoculum control 1	7.5
Inoculum control 2	7.5
Reference control 1	7.7
Reference control 2	7.7
Test item 1	7.5
Test item 2	7.6
Test item 3	7.6
Toxic control	7.6

Individual Daily Values for Cumulative Carbon Dioxide Production

	Test item group			Reference control group		Inoculum control group		Toxicity Control group
Time in d	Replicate 1	Replicate 2	Replicate 3	Replicate 1	Replicate 2	Replicate 1	Replicate 2	Replicate 1
	in mg/1.5L per vessel
0	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
2	16.2	15.3	18.8	39.3	30.8	9.08	7.98	31.1
5	33.6	44.8	23.2	34.9	36.0	25.0	18.4	34.4
7	16.8	15.4	15.1	19.5	18.8	13.8	9.63	18.0
9	9.49	8.53	12.7	8.80	13.3	7.56	7.84	10.3
15	5.36	8.80	16.2	15.4	24.5	16.1	12.9	22.1
23	19.7	25.3	35.3	11.3	19.1	14.3	12.2	15.5
28	15.1	16.8	17.2	6.46	8.66	6.19	6.88	6.74
29	10.3	6.33	6.19	3.58	5.23	3.30	6.33	5.91
					Cumulative: after 28 d	92.0	75.8
					Mean	82.8
					Cumulative: in mg/L	61.3	50.6
					Mean	55.2

Percentage Degradation

	Degradation^1 in %
Time in d	Test item group			Reference control group		Toxicity control group
	Replicate 1	Replicate 2	Replicate 3	Replicate 1	Replicate 2	Replicate 1
0	0	0	0	0	0	0
2	9	8	13	37	27	27
5	24	36	14	53	44	43
7	30	41	19	63	53	50
9	32	42	25	64	60	54
15	32	42	27	65	72	63
23	41	58	55	65	80	67
28	52	70	68	65	83	67
Mean (28 d)	62			74

After purging carbon dioxide overnight:

29	58	72	69	65	83	68
Mean (29 d)	66			74

¹ Corrected for the mean CO₂ production of the inoculum control group

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The ready biodegradability of IPEMA was assessed with the CO2 Evolution Test according to OECD Guideline 301 B. IPEMA was tested at a nominal concentration of 15 mg carbon/L.
The following biodegradation was determined at the end of the 28-d period:
IPEMA (test item): 62 %
Sodium benzoate (reference item): >60 %
Since the pass value of > 60 % was reached for the test item within 28 d, but not within the 10-d window (i.e. within 10 d from the time when biodegradation first reached 10 %) IPEMA cannot be considered to be readily biodegradable according to OECD guideline 301 B.
The test performance of the toxicity control group did not reveal any potential transient inhibition of microbial activity that may have interfered with the biodegradation of the test item under the conditions of this test. The validity criteria were met by the test.

Description of key information

IPEMA cannot be considered to be readily biodegradable according to OECD guideline 301 B.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater