Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 04, 2017 to October 30, 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)

Version / remarks:

Adopted 17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Communities, L 383 A, Part C.4-D, Biodegradation: Determination of ready biodegradability – Manometric respirometry. 29 December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

Activated sludge was obtained from Totnes Sewage Treatment Works, Totnes, Devon, UK on 23 August 2017. This works treats sewage of predominantly domestic origin. At the laboratory, the activated sludge was kept aerated at room temperature and the pH maintained at 7.0 ± 1.0. Six days prior to the exposure start the activated sludge was centrifuged, washed and re suspended in the mineral medium and the solids concentration determined. This sludge was then diluted in medium, added to test bottles and stirred until required for use. The seeded mineral medium was pre-conditioned for six days to reduce the blank oxygen uptake readings in the test.

Duration of test (contact time):

ca. 49 d

Initial conc.:

ca. 100 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test apparatus
The measurement of oxygen uptake was conducted in the Oxitop™ respirometers (Wissenschaftlich-Technische Werkstätten, GmbH, Germany). Each individual unit consisted of a dark glass 500 mL bottle with an Oxitop™ bottle top containing a piezo resistive electronic pressure sensor. Bottles were situated on a magnetic stirrer in a constant temperature incubator. Carbon dioxide produced by microbial respiration was absorbed by potassium hydroxide solution (KOH) placed in a seal cup in the neck of each bottle, and the oxygen taken up was measured as a decrease in pressure. The Oxitop™ controller collected the pressure values from the measuring tops and calculated the BOD (as mg/L).

Reference substance:

benzoic acid, sodium salt

Remarks:

100 mg/L

Test performance:

Validity Criteria
The validity requirements of the OECD guideline state:
·  The difference between extremes of replicate biodegradation values should be less than 20% at the end of 10-day window, at the plateau or at the end of the test;
·  The positive control should achieve > 60% biodegradation by Day 14. 
·  The oxygen consumption of the inoculum blank should not exceed 60 mg/L in 28 d.
Mean oxygen uptake of the inoculum blank was below 60 mg/L, as required in the OECD guideline. The difference between replicate test substanceextremes was < 20% after the 10 d window, at plateau, and on day 28. Sodium benzoate reached a mean 74% biodegradation by Day 14. The average of the mean oxygen consumed in the inoculum blanks was 6.9 mg/L after 28 d and 10.0mg/L after 49 d. Therefore, this test has satisfied all the validity criteria.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test substance

Value:

ca. 49

Sampling time:

28 d

Remarks on result:

other: not readily biodegradable

Parameter:

% degradation (O2 consumption)

Value:

61

Sampling time:

49 d

Remarks on result:

other: inherently biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Toxicity control

Value:

ca. 37

Sampling time:

14 d

Remarks on result:

other: >25% Biodegradability confirmed that the test substance is assumed not to be inhibitory at used concentration

Details on results:

pH and temperature measurements
At the end of the 49 d test period, the pH values ranged from 6.5 to 6.7 in the test substance bottles and from 6.4 to 6.6 in the toxicity control bottles. The inoculum blank and sodium benzoate bottles remained running until day 60 as these were shared controls with other studies, on day 60 the pH values were 7.3 in the inoculum blank bottles and ranged from 6.7 to 7.0 in the sodium benzoate bottles. Temperature measurements recorded in several of the test bottles on day 0 and all bottles on days 42 or 60 (as appropriate) indicated the temperature was within the range 22 ± 1ºC. Continuous monitoring of the incubator temperature showed it to have remained within the range 22 ± 1ºC throughout the study.

Results with reference substance:

Sodium benzoate did attain a mean level of biodegradation of 81% after 49 d, and the results showed good replication. More than 60% degradation was achieved within the 10 d window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms.

Results

Theoretical oxygen demand (ThOD)

The ThOD of sodium benzoate was calculated as 1.67 g O₂/g of substance, using the formula given in the OECD guideline. Biodegradation of sodium benzoate has been calculated on the basis of calculated ThOD.

Chemical oxygen demand (COD)

The measured COD value of test substance was 2.87 g O₂/g of substance, and its biodegradation was calculated on the basis of measured COD.

Biochemical oxygen demand (BOD)

The results for the reference substance did show that sodium benzoate attained a mean level of biodegradation (based on the BOD:ThOD ratio) of 81% after 49 d, and also showed a good replication. More than 60% degradation was achieved within the 10 d window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms.

Biodegradation results: Reference substance

Day	Sodium benzoate (blank corrected) BOD (mg/L)			BOD^a (g O2/g)			Biodegradation^b (%)
	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3	Mean
5	96.9	111	113	0.97	1.11	1.13	58	66	68	64
10	107	124	129	1.07	1.24	1.29	64	74	77	72
15	111	134	134	1.11	1.34	1.34	66	80	80	75
20	110	138	133	1.10	1.38	1.33	66	83	80	76
25	112	137	135	1.12	1.37	1.35	67	82	81	77
30	114	139	139	1.14	1.39	1.39	68	83	83	78
35	116	141	141	1.16	1.41	1.41	69	84	84	79
40	115	143	140	1.15	1.43	1.40	69	86	84	80
45	115	149	143	1.15	1.49	1.43	69	89	86	81
49	114	148	145	1.14	1.48	1.45	68	89	87	81

BOD: Biological Oxygen Demand

^a BOD (g O₂/g) =     Blank corrected biodegradation (mg/L)         

                             Sodium benzoate concentration (100 mg/L)

^b Biodegradation (%) =Biological Oxygen Demand (g O₂/g)× 100

                                                ThOD (1.67 g O₂/g)

The following phases (approximate) were observed:

Phase	Time period
Lag phase	Days 0 to 1
Degradation phase	Days 1 to 10
10-day window	Days 1 to 11

The results for test substance did show that it attained a mean level of biodegradation (based on the BOD:COD ratio) of 49% after 28 d and 61% after 49 d, and also showed a good replication. Less than 60% degradation was achieved within 28 d, so test substance could not be classified as readily biodegradable.

Biodegradation results: Test substance

Day	Test substance (blank corrected) BOD (mg/L)			BOD^a (g O2/g)			Biodegradation^b (%)
	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3	Mean
5	37.8	35.0	32.2	0.38	0.35	0.32	13	12	11	12
10	73.2	67.5	59.1	0.73	0.68	0.59	25	24	21	23
15	106	103	77.2	1.06	1.03	0.77	37	36	27	33
20	130	124	90.2	1.30	1.24	0.90	45	43	31	40
25	143	135	118	1.43	1.35	1.18	50	47	41	46
30	165	139	145	1.65	1.39	1.45	57	48	51	52
35	178	144	155	1.78	1.44	1.55	62	50	54	55
40	188	149	160	1.88	1.49	1.60	66	52	56	58
45	196	154	165	1.96	1.54	1.65	68	54	57	60
49	198	156	170	1.98	1.56	1.70	69	54	59	61

BOD:   Biological Oxygen Demand

^a Biological oxygen demand (g O₂/g) =Blank corrected biodegradation (mg/L)

                                               Substance concentration (100 mg/L)

^b Biodegradation (%) =Biological Oxygen Demand (g O₂/g) × 100

                                    COD (2.87 g O₂/g)

The following phases (approximate) were observed:

Phase	Time period
Lag phase	Days 0 to 4
Degradation phase	Days 4 to 34

The mean toxicity control degradation achieved on Day 14 was 37% (based on combined ThOD/COD), as this was > 25% the test substance was assumed not to be inhibitory at this concentration. 

Day	Toxicity control (blank corrected) BOD (mg/L)			BOD^a (g O2/g)			Biodegradation^b (%)
	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3	Rep 1	Rep 2	Rep 3	Mean
5	108	113	139	0.54	0.57	0.70	24	25	31	27
10	126	132	183	0.63	0.66	0.92	28	29	41	33
15	151	154	215	0.76	0.77	1.08	33	34	48	38
20	169	186	237	0.85	0.93	1.19	37	41	52	43
25	188	211	258	0.94	1.06	1.29	41	47	57	48
30	215	227	283	1.08	1.14	1.42	48	50	63	54
35	237	237	296	1.19	1.19	1.48	52	52	65	56
40	250	241	303	1.25	1.21	1.52	55	53	67	58
45	264	244	309	1.32	1.22	1.55	58	54	68	60
49	271	252	314	1.36	1.26	1.57	60	56	69	62

 BOD: Biological Oxygen Demand

^a Biological oxygen demand (g O₂/g) = Blank corrected biodegradation (mg/L) 

                                            Toxicity control concentration (200 mg/L)

^b Biodegradation (%) =Biological Oxygen Demand (g O₂/g) × 100

                                     Combined ThOD/COD (2.27 g O₂/g)

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Under the study conditions, the test substance was classified as inherently biodegradable (49% after 28 d and 61% after 49 d).

Executive summary:

A study was conducted to determine the biodegradability of the test substance, C16-18 AMP, according to OECD Guideline 301F and EU Method C.4-D (Manometric respirometry test), in compliance with GLP. Following pre-conditioning, the test bottles (activated sludge inoculum in mineral medium) were set up for the test according to the experimental design. Positive control (sodium benzoate at 100 mg/L), test substance (100 mg/L) and toxicity controls (test and reference substances, both at 100 mg/L) were prepared in triplicate, while inoculum blanks were prepared in duplicate. Additional single replicate test substance and toxicity control bottles were prepared for determination of pH at Day 0, so that undissolved test substance was not removed by the pH probe. Oxygen uptake was recorded automatically every 240 min during the 49 d experimental period. Oxygen uptake values were corrected for the inoculum blank and biodegradation was calculated as a percentage of the chemical oxygen demand (COD) of the test substance and as a percentage of the theoretical oxygen demand (ThOD) of the reference substance. The COD of test substance was determined using spectrophotometer for evaluation. Test substance attained a mean level of biodegradation (based on the BOD:COD ratio) of 49% after 28 d and 61% after 49 d. The test substance results showed good replication and exhibited the potential for rapid degradation. Sodium benzoate attained a mean level of biodegradation (based on the BOD:ThOD ratio) of 81% after 49 d and also showed a good replication. More than 60% degradation was achieved within the 10 d window, as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. The mean toxicity control degradation achieved on Day 14 was 37% (based on combined ThOD/COD). Since this was > 25%, the test substance was assumed not to be inhibitory at this concentration. The test satisfied all guideline validity criteria. Under the study conditions, the test substance was not readily biodegradable but could be classified as inherently biodegradable (Scymaris, 2017).