Ammonium formate

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:

2019-07-08 to 2019-12-11

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:

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 sewage treatment plant, 31137 Hildesheim, Germany
- Preparation of inoculum for exposure:
The following incubation vessels were prepared:
- two for the inoculum control:(C 1 , C 2 )
- one for the functional control: (R 1 )
- two for the test item concentration: (P 1 , P 2 )
- one for the toxicity control: (T 1 )
The necessary amounts of ultrapure water, mineral salts medium and inoculum were placed in each incubation vessel. The vessels were aerated for 24 h with CO 2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH) 2 solution.
The test item and reference item were weighed out. The test item was weighed out into small beakers. A defined amount of ultrapure water was added to the test item. The test item dispersions and the reference item were transferred to the respective incubation vessels with ultrapure water.
- Pretreatment: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration with CO 2 free air for two days before test start. Further treatment see section ‘preparation of the test vessels’. 4.0 mL/L of this mixture were used to initiate inoculation (25.1 mg/L dw).
- Concentration of sludge:
- Initial cell/biomass concentration: Approx. 1E+07 - 1E+08 CFU/L

Duration of test (contact time):

28 d

Initial conc.:

55 mg/L

Based on:

test mat.

Initial conc.:

10.5 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO 2 Evolution Test
- Test temperature: Nominal 22 ± 2 °C, actually measured 20 – 23 °C
- pH: Between 7.16 and 7.62 at day 28
- pH adjusted: no
- CEC (meq/100 g):
- Aeration of dilution water: yes 30 - 100 mL/min
- Suspended solids concentration: 25.1 mg/L dw
TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: two for the test item, two for inoculum control, one for functional control and one for toxicity control
- Measuring equipment: Determination of CO 2 was carried out by titration subsequent to complete adsorption of the released CO 2 in an alkaline solution (0.0125 mol/L Ba(OH) 2 ).Back titration of the residual Ba(OH) 2 with 0.05 N HCl.
Equipment
pH-Meter, Multi 350i, WTW
Thermohygrograph, LUFFT
Analytical balance, SARTORIUS Balance, KERN
Dispensette, BRAND
Digital Buret, continous RS, VITLAB
Medo compressor, FA. REBIE
Magnetic stirrer Mono, VARIOMAG
Multipette X-Stream, EPPENDORF
Various Pipettes
SAMPLING
- Sampling frequency: Back titration of the residual Ba(OH) 2 with 0.05 N HCl was carried out three times a week during the first ten days and thereafter
twice weekly.On day 28 the pH of all solutions was measured prior to acidification.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, two vessels
- Toxicity control: Yes, one vessel

STATISTICAL METHODS:
The theoretical production of carbon dioxide (ThCO 2 ) of the test item and functional control is calculated by the carbon content (1) and the molecular formula (2), respectively.
ThCO2 [mgCO2/mg] = 3.67 · TOC [mgC/mg test item] (1)

ThCO2 [mgCO2/mg] = C-Atoms x molecular weight of CO2/molecular weight of test item (2)
The produced CO2 will be calculated by (3):

1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2 (3)
The net amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the inoculum controls. The biodegradation will be calculated from the ratio theoretical CO2 production to net CO2 production in the following equation (4):

Degradation [%] = netCO2 x 100/ThCO2[mgCO2/3L]

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Replicate 1

Value:

14

Sampling time:

4 d

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Replicate 2

Value:

15

Sampling time:

4 d

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Replicate 1

Value:

75

Sampling time:

8 d

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Replicate 2

Value:

74

Sampling time:

8 d

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Replicate 1

Value:

101

Sampling time:

28 d

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

Replicate 2

Value:

94

Sampling time:

28 d

Details on results:

The mean of replicates reached the 10 % level (beginning of biodegradation) within 4 days. Both test item replicates reached the 60 % pass level within 8 days. The mean biodegradation on day 28 was 98 %. The 10-day window was fulfilled for the replicates: The mean of replicates passed the 10 % threshold on day 3. 10 days later, the mean biodegradation of replicates was > 60 %.

Results with reference substance:

The adaptation phase of the functional control changed within 1 day into the degradation phase (degradation  10 %). The course of the degradation was fast and the functional control reached the pass level of 60 % within 6 days and a maximum biodegradation of 88 % on day 28. The validity criterion degradation  60 % after 14 days was fulfilled.

CO₂-Production and Biodegradation in the Inoculum Control, the Functional Control and the Toxicity Control

Study Day	Date	Inoculum Control	Functional Control			Toxicity Control
		[mg CO2/3 L]	[mg CO2/3 L]		Degr.	[mg CO2/3 L]		Degr.
		mv	Gross	Net Sum	[%]	Gross	Net Sum	[%]
1	2019-09-20	6.2	20.4	14.2	11	17.1	10.9	4
4	2019-09-23	16.0	53.4	51.6	40	53.8	48.7	20
6	2019-09-25	12.4	40.9	80.1	63	53.8	90.1	37
8	2019-09-27	12.2	24.2	92.1	72	53.1	131.0	54
11	2019-09-30	12.5	19.5	99.1	78	53.2	171.7	71
15	2019-10-04	13.2	19.1	105.0	82	45.0	203.5	84
18	2019-10-07	10.8	14.2	108.4	85	14.0	206.7	85
22	2019-10-11	9.4	11.6	110.6	87	10.8	208.1	86
25	2019-10-14	6.3	7.5	111.8	87	7.5	209.3	86
28	2019-10-17	9.1	6.8	111.8	87	9.4	209.6	86
29*	2019-10-18	6.8	6.8	112.1	88	5.7	209.9	86

Degr. = degradation; mv = mean value; *) results of last two gas wash bottles

CO₂-Production and Biodegradation in the Inoculum Control and Test Item Replicates

Study Day	Date	Inoculum Control	Test Item
			Replicate 1			Replicate 2
		[mg CO2/3 L]	[mg CO2/3 L]		Degr.	[mg CO2/3 L]		Degr.
		mv	Gross	Net Sum Sum Sum	[%]	Gross	Net Sum	[%]
1	2019-09-20	6.2	7.4	1.2	1	8.3	2.1	2
4	2019-09-23	16.0	31.4	16.6	14	30.9	17.0	15
6	2019-09-25	12.4	49.0	53.2	46	52.0	56.6	49
8	2019-09-27	12.2	46.0	87.0	75	40.8	85.2	74
11	2019-09-30	12.5	28.7	103.2	89	25.1	97.8	85
15	2019-10-04	13.2	19.7	109.7	95	17.2	101.8	88
18	2019-10-07	10.8	12.9	111.8	97	13.3	104.3	90
22	2019-10-11	9.4	10.5	112.9	98	10.9	105.8	92
26	2019-10-14	6.3	7.9	114.5	99	7.6	107.1	93
28	2019-10-17	9.1	10.6	116.0	100	10.1	108.1	94
29*	2019-10-18	6.8	7.8	117.0	101	7.3	108.7	94

Degr. = degradation; mv = mean value; *) results of last two gas wash bottles

pH-Values on Day 28

Inoculum Control		Functional Control	Test Item		Toxicity Control
No. 1	No. 2	No.1	No. 1	No. 2	No. 1
7.53	7.51	7.62	7.15	7.16	7.21

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In the present test the biodegradability of Ammonium Formate was determined according to OECD Guideline 301B (Modified Sturm Test). The test item (55 mg/L corresponding to 10.5 mg/L TOC), the inoculum control, a functional control and a toxicity control were incubated under aerobic conditions for 28 days. Subsequently the CO2 Evaluation was measured by titration. The test item exhibited a degradation of 15% on day 4 and on day 15 a degradation 75%, at the end of the test on day 28 the degradation was 101%. Thus, Ammonium formate is considered to be readily biodegradable.

Executive summary:

The biodegradation of Ammonium formate was investigated over a 28-day period in a CO2 Evolution Test according to OECD guideline 301 B (1992). The test medium was inoculated with non-adapted activated sludge from a sewage treatment plant mainly fed with municipal wastewater. The rate of degradation was monitored by measuring the carbon dioxide produced over the 28-d period.

The test item was tested with a concentration of 55 mg/L in duplicates, corresponding to a carbon content (TOC) of 10.5 mg C/L in the test vessels.

The biodegradation of the test item was followed by titrimetric analyses of the quantity of CO2 produced by the respiration of bacteria. The degradation was finished on day 28 by acidification, the last titration was made on day 29. After the soluble CO2 was turned out over a period of 24 h.

The percentage CO2 production was calculated in relation to the theoretical CO2 (ThCO2) of the test item. The biodegradation was calculated for each titration time.

The 10 % level (beginning of biodegradation of the test item) was reached within an adaptation phase of 4 days. The biodegradation was rapid, the pass level of 60 % was reached already on day 8. The mean biodegradation came to a maximum of 98 % after 28 days.

To check the activity of the test system sodium acetate was used as functional control. The percentage of degradation of the functional control reached the pass level of 60 % after 6 days.

In the toxicity control containing both test and reference item a biodegradation rate of 84 % occurred within 15 days and came to a maximum of 88 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

The test item Ammonium formate is readily biodegradable in this CO2 evolution test and fulfills the 10 d window criterion. The substance is not inhibitory to microorganisms at a concentration of 55 mg/L.

 

Description of key information

The test item is readily biodegradable based on the results from a test performed according to OECD Guideline 301B (Modified Sturm Test).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater