1-nitroguanidine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April - May 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Remarks:

Quality assurance statement: The study was conducted in accordance with SN EN 45001 (in-house quality standard).

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: activated sludge from the aeration tank of a municipal biological waste water treatment plant, not adapted, not pre-conditioned
- Laboratory culture:
- Method of cultivation:
- Storage conditions:
- Storage length:
- Preparation of inoculum for exposure:
- Pretreatment:
- Concentration of sludge: 0.2 g/l dry matter in the final mixture
- Initial cell/biomass concentration:
- Water filtered: yes/no
- Type and size of filter used, if any:

Duration of test (contact time):

28 d

Initial conc.:

521 mg/L

Based on:

test mat.

Initial conc.:

48.6 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

calculated as % ThCO2

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST SYSTEM AND TEST CONDITIONS

1. Test unit:
- 1200 mL closed glass bottle containing a total volume of test solution of 600 mL
- aerated with CO2-free air and fitted to gas-absorption bottles containing 120 mL of 0.05 M NaOH
- c(O2) > 6 mg/L

2. Test medium:
- aerobic mineral salt medium prepared with double distilled water (conductivity < 1.5 µS/cm; DOC < 0.3 mg/L)
- for details see table 1

3. Feed:
- None, test substance or procedure control as sole organic carbon sources

4. Incubation:
- temperature-controlled dark room (22 +/- 0.5 °C)


TEST PROCEDURE:
- prior to the test sludge was washed twice with tap water
- the test material was diluted with mineral salts medium to give a final DOC concentration of about 50 mg/L
- number of test flasks for each test series:
* test suspension (T) 2 replicates: containing activated sludge + test medium + test substance
* inoculum blank (B) 2 replicates: containing activated sludge + test medium
* procedure control (R) 1 replicate: containing activated sludge + test medium + diethylenglycol as ready biodegradable reference substance
- pH-value was checked peridically and adjusted to pH 6.5-8.0 with NaOH or H2SO4, if necessary
- elimination of the test material was followed by DOC determinations at regular intervals; first samples were analyzed at the beginning and 3 h after the start of the test
- the trapped CO2 was determined as inorganic carbon (IC)


SAMPLING
- Sampling frequency: after 0, 0.125, 1, 3, 7, 10, 14, 16, 21, 24, 27, and 28 days
- Sampling method: see "Details on analytical methods"

Reference substance:

diethylene glycol

Remarks:

49.9 mg/L (as DOC)

Preliminary study:

No preliminary study

Test performance:

No unusual observations during testthat could have affected the results.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: calculated as % ThCO2

Details on results:

Based on the data of the individual DOC determinations (see table 2) no biodegradation of nitroguanidine (moistened) was obserevd after 28 days.
No significant elimination of the test substance due to adsorption to the activated sludge, on the glass surface or other physico-chemical processes was found as determined by means of DOC measurements 3h after the start of the test.
No biodegradation of nitroguanidine (moistened) based on CO2 evaluation and calculated as % ThCO2 was observed. The extent of mineralization of the procedure control with diethylenglycol was 81 % based on ThCO2 (see table 3).

Results with reference substance:

The positive control, diethylenglycol, showed 99 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions.

Table 2: DOC concentrations of the test suspension, inoculum blank and procedure control and calculation of degradation data

	Inoculum Blank (B)*	Procedure control with diethylenglycol (R)			Test suspension with test material (T)
Time (days)	DOC (mg/L)	DOC (mg/L)	DOC net. (mg/L)	Degradation (%)	DOC (mg/L)	DOC net. (mg/L)	Degradation (%)
0	0.6	50.7	50.2	-	50.3	49.7	-
0.125	1.2	52.3	51.1	0	50.5	49.3	0
1	1.5	46.9	45.4	11	51.0	49.5	0
3	1.9	39.8	37.9	26	52.2	50.3	-2
7	3.0	4.0	1.0	98	52.7	49.6	-1
10	2.8	3.7	0.9	98	52.0	49.2	0
14	2.7	3.4	0.7	99	53.1	50.4	-2
16	3.0	3.5	0.5	99	52.6	49.6	-1
21	3.8	4.0	0.2	100	54.4	50.6	-3
24	2.3	2.6	0.3	100	51.0	48.7	1
27	2.4	2.4	0.0	100	51.5	49.1	0
28	2.6	2.8	0.2	100	52.0	49.3	0

 

Table 3: IC concentrations, calculated from the concentrations in the gas absorption bottles, of test suspension, inoculum blank and procedure control and corresponding degradation data

Time (days	Test suspension IC (mg/L)	Procedure control IC (mg/L)	Inoculum blank (mg/L)	Biodegradation Test suspension (% ThCO2)	Biodegradation Procedure control (%ThCO2)
0	nd	nd	nd	-	-
7	17.3	35.8	17.7	-1	36
14	33.9	71.6	30.9	6	82
21	37.1	78.3	37.3	0	82
28	37.2	76.9	36.3	2	81

 

Validity criteria fulfilled:

yes

Remarks:

The test was considered valid, since the degradation of the reference compound reached more than 70 % within 14 days of incubation.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Based on these results niitroguanidine (moistened) is not biodegradable under the conditions of OECD guideline no. 302, since no degradation was attained after 28 days of contact time.

Executive summary:

The biodegradability of nitroguanidine (moistened) exposed to activated sludge of a municipal sewage treatment plant was investigated under aerobic static conditions.

 

Based on the data of the individual DOC determinations no biodegradation of nitroguanidine (moistened) was observed after 28 days.

 

No significant elimination of the test substance due to adsorption to the activated sludge, on the glass surface or other physico-chemical processes was found as determined by means of DOC measurements 3h after the start of the test.

 

The positive control, diethylenglycol, showed 99 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions. The test was considered valid, since the degradation of the reference compound reached more than 70 % within 14 days of incubation.

 

No biodegradation of nitroguanidine (moistened) based on CO₂ evolution and calculated as % ThCO₂ was observed. The extent of mineralization of the procedure control with diethylenglycol was 81 % based on ThCO₂.

 

Based on these results nitroguanidine (moistened) is not biodegradable under the conditions of OECD guideline no. 302, since less than 20 % degradation was attained after 28 days of contact time.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1982

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: not reported

Principles of method if other than guideline:

Incubation of nitroguanidine solution with activated sludge microorganisms; Measurement (HPLC) of the concentration of nitroguanidine over time

GLP compliance:

not specified

Specific details on test material used for the study:

Test substance supplier: Radford Army Ammunition Plant.

Oxygen conditions:

aerobic/anaerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Key result

Parameter:

% degradation (test mat. analysis)

Value:

0

Remarks on result:

other: aerobic conditions

Validity criteria fulfilled:

not applicable

Interpretation of results:

other: Nitroguanidine was not susceptible to aerobic biodegradation in activated sludge. Nitroguanidine was co-metabolized by anaerobic sludge microorganisms to nitrosoguanidine after acclimation.

Conclusions:

Nitroguanidine was not susceptible to aerobic biodegradation in activated sludge. Nitroguanidine was co-metabolized by anaerobic sludge microorganisms to nitrosoguanidine after acclimation.

Executive summary:

Nitroguanidine was tested for biodegradability under aerobic and anaerobic conditions. Nitroguanidine was not susceptible to aerobic biodegradation in activated sludge. Nitroguanidine was cometabolized by anaerobic sludge microorganisms to nitrosoguanidine after acclimation.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 1986 - April 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Biotransformation was assessed using microorganisms acclimated to nitroguanidine.
For kinetic experiments, the cells were resuspended in 250 mL of phosphate buffer to make a high population cell suspension, and 80 mL was dispensed into 150 mL bottles. The NQ was added to the bottles from the aqueous stock solution and incubated with or without the addition of nutrient broth and yeast extract.

GLP compliance:

not specified

Specific details on test material used for the study:

Test substance supplier: Aldrich Chemical Co
Water Content: 25%

Oxygen conditions:

aerobic/anaerobic

Inoculum or test system:

natural water: freshwater

Details on inoculum:

- Source of inoculum: microorganisms from pond B of SAAP (Sunflower Army Ammunition Plant)
- Sampling: Pond B water was collected and shipped overnight to SRI under ice
- Laboratory culture:
* 250 ml pond B water + 1 g/l nutrient broth, 1 g/l yeast extract, 0.5 g/l potassium phosphate buffer (pH 7.0), and 10 ppm nitruguanidine;
* incubation at 20-25 °C with periodic shaking by hand;
* removal of 5 ml solution at various intervals and determination of nitroguanidine (HPLC);
* when > 70 % of nitroguanidine was degraded, the microorganisms were transferred to a medium containing the same components as described above
* after several transfers, the miccroorganisms were grown in deionized water medium containing 200 ppm nutrient broth, 200 ppm yeast extract, 0.5 g/l phosphate buffer, and 20 ppm nitroguanidine solution in 1 L contained in a 2 L flask loosely covered with aluminium foil and statically incubated;
* typically, 90 % of nitroguanidine was transformed in two days.
- Method of cultivation:
- Storage conditions:
- Storage length:
- 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):

10 h

Initial conc.:

2.1 other: ppm

Based on:

test mat.

Initial conc.:

8.5 other: ppm

Based on:

test mat.

Initial conc.:

11.5 other: ppm

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

- After 2 days of incubation of microorganisms in water medium, the broth was centrifuged at 4000 x G for 10 min., washed with phosphate buffer, and recentrifuged.
- The cells were resuspended in 250 ml of phosphate buffer to make a high population cell suspension, and 80 ml was dispensed into 150-ml bottles.
- Nitroguanidine was added to the bottles from an aqueous stock solution and incubated with or without the addition of nutrient broth (NB) and yeast extract (YE).
- Periodically, the bottles were shaken by hand and a 5 mL sample was removes, placed in a vial containing 1 drop of 2 % HgCl2 solution, and analyzed by HPLC.
- Viable cell counts were made with Difco triptic soy broth agar incubated in BBL canopy pack microaerophilic system jar.
The preliminary results showed nearly identical plate counts for the aerobic and microaerophilic plates, indicationg that most of the bacteria are facultative.

Key result

Parameter:

% degradation (test mat. analysis)

Value:

50

Sampling time:

85 d

Remarks on result:

other: cometabolic biotransformation, low nutrient conditions; results were calculated from the second-order rate constant of nitroguanidine transformation

Key result

Parameter:

% degradation (test mat. analysis)

Value:

50

Sampling time:

30 d

Remarks on result:

other: cometabolic biotransformation, high nutrient conditions; results were calculated from the second-order rate constant of nitroguanidine transformation

Details on results:

Nitroguandine was biodegradable in dependence on the nutrient concentration (nutrient broth, yeast).

The different nitroguanidine concentration experiments were performed at different times and the experiments were performed over 10 hour periods. The initial aerobic plate count was used for the second-order biotransformation rate constant calculation. The plots clearly indicate the rate dependence on extra organic nutrients, which may serve as the energy source for the biotransformation and just as growth substrate. The biotransformation by cells without additional nutrients may rely on stored energy to effect the transformation.

The first- and second-order rate constants determined for the biotransformation as a function of nitroguanidine concentration and organic nutrient are shown in the table below:

Nitroguanidine [ppm]	NB + YE [ppm each]	k^1b [hr^-1]	X [Org/mL]	kb2 [mL org^-1­hr^-1]
2.1	0	0.033	8.40 x 10^7	4.0 x 10^-10
	20	0.102	8.40 x 10^7	1.2 x 10^-9
	50	0.192	8.40 x 10^7	2.3 x 10^-9
8.5	0	0.078	1.67 x 10^8	4.6 x 10^-10
	20	0.303	1.67 x 10^8	1.8 x 10^-9
	50	0.341	1.67 x 10^8	2.0 x 10^-9
11.5	0	0.028	1.01 x 10^8	2.8 x 10^-10
	50	0.265	1.01 x 10^8	2.6 x 10^-9

 

The average second-order rate constant (k_b2) for microbes was 3.8 (+/- 0.9) x 10^-10mL org^-1hr^-1without additional nutrients; 1.5 (+/- 0.4) x 10^-9mL org^-1hr^-1with 20 ppm additional nutrients; 2.3 (+/- 0.3) x 10^-9mL org^-1hr^-1with 50 ppm additional nutrients.

From the second order rate constants it is possible to estimate nitroguanidine persistence under specific environmental conditions. In a quiescent water containing 1 x 10⁶org/ml and a low nutrient level, a first-order rate constant can be calculated and the half-life determined as shown in the following equations:

k¹_b= k_b2* X = (3.8 x 10^-10) (1 x 10⁶) = 3.8 x 10^-4 hr^-1­

half-life = ln2/k¹_b= 0.69/3.8 x 10^-4 = 2038 h = 85 days

In a pond bottom containing decomposed organic matter, the microbial population may be in the range of 1 x 10⁷org/mL. Assuming 100 ppm organic matter, the first-order rate constant will be:

K¹_b= (2.3 x 10^-9) (1 x 10⁷) = 2.3 x 10^-2­hr^-1, and the half-life will be 30 days.

 

Validity criteria fulfilled:

not applicable

Interpretation of results:

other: biodegradation as a co-metabolic process

Conclusions:

The biotransformation was shown to be a co-metabolic process in which the organisms could degarde nitroguanidine only in the presence of other organic nutrients (such as nutrient broth) and nor as a sole carbon and energy source.

Executive summary:

From this study it becomes clear that the microbial persistence of nitroguanidine and the nitroguanidine biotransformation rate constants are dependent on the environment with regard to the oxygen and nutrient concentration. Biotransformation of nitroguanidine can occur under both aerobic and anearobic conditions. The biotransformation was shown to be a co-metabolic process in which the organisms could degrade nitroguanidine only in the presence of other organic nutrients (such as nutrient broth) and nor as a sole carbon and energy source.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

other: Handbook data

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Principles of method if other than guideline:

examination of nitroguanidine degradation (aerobic conditions); no details reported

GLP compliance:

not specified

Specific details on test material used for the study:

Test substance supplier not specified

Oxygen conditions:

aerobic

Inoculum or test system:

not specified

Remarks on result:

not measured/tested

Details on results:

No details reported

No details reported (secondary literature).

According to the literature cited (Rosenblatt et al., Organic explosives and related compounds, 1991), Nitroguanidine is not susceptible to aerobic biodegradation.

Validity criteria fulfilled:

not applicable

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Nitroguanidine is not susceptible to aerobic biodegradation.

Executive summary:

According to the literature cited (Rosenblatt et al., Organic explosives and related compounds, 1991), nitroguanidine is not susceptible to aerobic biodegradation.

Description of key information

The inherent biodegradability of nitroguanidine was determined using OECD method 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test). The biodegradability of nitroguanidine (moistened) exposed to activated sludge of a municipal sewage treatment plant was investigated under aerobic static conditions. Nitroguanidine was tested at a concentration of 48.9 mg/L DOC. The reference substance diethylene glycol was tested at a concentration of 49.9 mg/L DOC. From the results of these tests it can be concluded that nitroguanidine is not biodegradable under aerobic conditions in standard tests but can be bio transformed co-metabolically by microorganisms after acclimation according to available literature data.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The inherent biodegradability of nitroguanidine was determined using OECD method 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test). The biodegradability of nitroguanidine (moistened) exposed to activated sludge of a municipal sewage treatment plant was investigated under aerobic static conditions. Nitroguanidine was tested at a concentration of 48.9 mg/L DOC. The reference substance diethylene glycol was tested at a concentration of 49.9 mg/L DOC.

 

Based on the data of the individual DOC determinations (OECD 302B), no biodegradation of nitroguanidine (moistened) was observed after 28 days.

No significant elimination of the test substance due to adsorption to the activated sludge, on the glass surface or other physico-chemical processes was found as determined by means of DOC measurements 3h after the start of the test.

The positive control, diethylene glycol, showed 99 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions. The test was considered valid, since the degradation of the reference compound reached more than 70 % within 14 days of incubation.

 

Results from the literature support the outcome of this OECD Guideline test. Literature results confirm that nitroguanidine is not biodegradable under aerobic conditions but indicates that nitroguanidine can be co-metabolically degraded by activated sludge microorganisms under anaerobic conditions after acclimation (Kaplan et al., 1982). This result is also supported by Spanggord et al., 1987, who were able to demonstrate that the biotransformation of nitroguanidine is a co-metabolic process in which the organisms could degrade nitroguanidine only in the presence of other organic nutrients (such as nutrient broth) and not as a sole carbon and energy source.

 

Therefore, it can be concluded that nitroguanidine is not biodegradable under aerobic conditions in standard tests but can be biotransformed co-metabolically by microorganisms after acclimation.