1-nitroguanidine

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sediment simulation testing

Data waiving:

other justification

Justification for data waiving:

the study does not need to be conducted because direct and indirect exposure of sediment is unlikely

other:

Transformation products:

no

Reference 2

Endpoint:

biodegradation in water: sewage treatment simulation testing

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

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, Radford

Radiolabelling:

no

Oxygen conditions:

aerobic/anaerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Aerobic cultures were inoculated with activated from the Marlborough Easterly sewage treatment plant (Marlborough, MA, USA).
- Anaerobic cultures were inoculated eith digest from the Nut Isalnd sewage treatment plant (Moston, MA, USA).
- The aerobic and anaerobic sludges contained < 0.1 % and 1.7 % total solids, respectively.
- Sludge samples were diluted 100-fold with 0.85 % KCl and filtered, and 0.5 mL was added to the culture flasks.

Duration of test (contact time):

3 mo

Initial conc.:

>= 50 - < 100 other: ppm

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

CULTURE CONDITIONS:
1. Aerobic batch cultures:
- incubated in 250 mL Erlenmeyer flasks each containing 100 mL of media at 30 °C on a New Brunswick G24 environmental incubator shaker

2. Anaerobic (unaerated) batch cultures and sterile controls:
- incubated at 37 °C in 250 mL Erlenmeyer flasks filled with media
- some anaerobic controls and incubations contained 0.05 % dithiothreitol as a reducing agent

3. New Brunswick Bio Flow Model C30 bench-top chemostats for continous culture:
- Aerobic and anaerobic conditions
- Media: either basal salts with nitrogen and glucose or nutrient broth (2 and 4 g/L)
- Retention time: 7 days
- Temperature: 30 °C
- Influent nitroguanidine concentration: 75-100 µg/mL

4. Anaerobic chemostats:
- Media: nutrient broth (2, 4 and 8 g/L), basal salts, basal salts with glucose, and basal salts with glucose and nitrogen
- Initial retention time: 7 days; later dropped to 4 and 2 days
- Initial nitroguanidine concentration: 50-100 µg/mL
- Chemostats were operated continously for up to 3 months at 37 °C

Reference substance:

not specified

Key result

Remarks on result:

not measured/tested

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

Proposed pathway of chemical and biological degradation of nitroguanidine:

1. Nitroguanidine -> Nitrosoguanidine

2. Nitrosoguanidine -> Cyanamide + Nitrosamide

3. a) Cyanamide -> Guanidine

3. b) Cyanamide -> Cyanoguanidien -> Melamine

4. Nitrosamid -> N₂ + H₂O

Validity criteria fulfilled:

not applicable

Conclusions:

Nitroguanidine was not susceptible to aerobic biodegradation in activated sludge, and it was stable under sterile reducing conditions. Nitroguanidine was co-metabolized by anaerobic sludge microorganisms to nitrosoguanidine after acclimation.

Executive summary:

Nitroguanidine was not susceptible to aerobic biodegradation in activated sludge, and it was stable under sterile reducing conditions. Nitroguanidine was cometabolized by anaerobic sludge microorganisms to nitrosoguanidine after acclimation. There was no further microbial reduction of nitrosoguanidine (no aminoguanidine, hydrazine or urea was detected in culture extracts). Nitrosoguanidine decomposed nonbiologicllay and formed cyanamide, cyanoguanidine, melamine, and guanidine. All products were identified by thin-layer chromatography and mass-spectroscopy. A pathway for the degradation of nitroguanidine is proposed.

Reference 3

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September 1982 - October 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

other: not reported

Principles of method if other than guideline:

literature review

GLP compliance:

not specified

Specific details on test material used for the study:

Test substance supplier: Sunflower AAP

Radiolabelling:

no

Oxygen conditions:

anaerobic

Inoculum or test system:

activated sludge, adapted

Key result

Remarks on result:

not measured/tested

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

This document is a literature review from 1982. Details on results like % degradation were not reported.

Validity criteria fulfilled:

not applicable

Conclusions:

Under anaerobic conditions nitroguanidine is biotransformed co-metabolically to nitrosoguanidine by microorganisms of activated sludge.

Executive summary:

Under anaerobic conditions nitroguanidine biotransforms to nitrosoguanidine. Microorganisms were those of activated sludge. Cultures required acclimatization, and biotransformation was cometabolic, with 4 g/L nutrient broth optimum. Biotransformation of nitroguanidine to nitrosoguanidine was observed with subsequent nonbiologically-mediated formation of the intermediates cyanamide, cyanoguanidine, melamine, and guanidine.

Reference 4

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

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. Nitroguanidine 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 Chemicla Co
Water content: 25%

Radiolabelling:

no

Oxygen conditions:

aerobic/anaerobic

Inoculum or test system:

natural water

Details on source and properties of surface water:

- Water from Pond B of Sunflower Armay Amunition Plant (SAAP), DeSoto, Kansas
- Storage conditions: under ice
- Storage length: samples were sent to laboratory by overnight express

Details on source and properties of sediment:

EPA-6:
- pH: 7.83
- CEC (meq/100g): 33.01
- Total N %: 0.097
- Organic carbon %: 0.72
- Sand %: 0.2
- Clay %: 68.6
- Silt %: 31.2

EPA-12:
- pH: 7.63
- CEC (meq/100g): 13.53
- Total N %: 0.214
- Organic carbon %: 2.33
- Sand %: 0.0
- Clay %: 35.4
- Silt %: 64.6

Soil sorption experiments were conducted seperatly from biotransformation experiments. > No contact to sediment during biodegradation experiments.

Details on inoculum:

- Source of inoculum: microorganisms were obtained from Pond B at SAAP and acclimated to nitroguanidine
- Method of cultivation: microorganisms were acclimated tonitroguanidine as follows: 1 g/L nutrient broth, 1 g/L yeast extract, 0.5 g/L potassium phosphate buffer (pH 7.0) and 10 ppm nitroguanidine were added to 250 mL of the Pond B water in a 500 mL Erlenmeyer flask. The water was incubated at 20 to 25 °C for several days, with periodic shaking by hand.
- At various intervals a 5 mL sample was removed, one drop of 2 % HgCl2 solution was added to inhibit biological activity, and the sample was analyzed by HPLC.
- When more than 70 % of nitroguanidine was degraded, the microorganisms were centrifuged and transferred to fresh medium.
- After several transfers, the microorganisms were grown in deionized water containing 200 ppm nutrient broth, 200 ppm yeast extract, 0.5 g/L phosphate buffer and 20 ppm nitroguanidine solution in 1L contained in a 2-L flask loosely covered with aluminium foil and statically incubated.
- Typically, 90 % of the nitroguanidine was transformed in 2 d. After 2 d of incubation, the broth was centrifuged at 4000 g for 10 min, washed with phosphate buffer and recentrifuged.

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 phosphare 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 removed, 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.

Test performance:

No unusual observations during test.

Key result

Compartment:

water

DT50:

85 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Aerobic conditions; estimation for a quiescient water body containing 1 x 10^6 cells/mL and a low nutrient level

Key result

Compartment:

water

DT50:

30 h

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Aerobic conditions; estimation for microbial population of 1 x 10^7 cells/mL, assuming 100 ppm organic matter.

Other kinetic parameters:

other: avarage second-order rate constant without additional nutrients: 3.8 (+/- 0.9) x 10^-10 ml/(cell*h)... (see attached file)

Transformation products:

yes

No.:

#1

Details on transformation products:

- The monitoring of the nitroguanidine biotransformation by HPLC indicated no new products that were resovable and detectable at 254 nm.
- N-Nitrosoguanidine and N-hydroxyguanidine would have been resolved and detected had they been formed and released to the solution.
- Lyophilization of a centrifuged and filtered suspension yielded a solid residue (pronicpally inorganic salts)
- When the residues were treated with BSTFA, cyanamide was identified by GC.MS (as compared to a reference spectrum) as the bis-trimethylsilyl derivative.
- Ion chromatographie analysis of the transformed solutions indicated no nitrite or nitrate.
- These data suggest a complete reductive pathway with the primary steps occuring rapidly within the cell to detoxify NQ and finally release cyanamide.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

- The aerobic biotransformation of nitroguanidine was found to occur slowly in natural waters and was accelerated by the presence of nutrient broth or yeast extract.
- Because the microorganisms transformed nitroguanidine by a co-metabolic process, nitroguanidine (NQ) was considered not to be a growth substrate.
- Under these conditions, a Monod kinetic analysis connot be applied.
- The transformation rates were approximated by a pseudo-first order rate expression (ln [NQ]/[NQ]o= -kb1 * t) using a high cell population and transformation periods in which the cell population remained constant.
- Biodegradation of nitroguanidine follows first-order kinetic behaviour and clearly indicates the rate dependence on extra organic nutrients, which may serve as an energy source for the transformation and not just as growth substrates.
- The biotransformation without additional nutrients may rely on stored energy to effect the transformation.
- Pseudo-first-order and second-order biotransformation rate constants determined for the biotransformation of nitroguanidine as a function of nitroguanidine concentration and organic nutrient concentration are shown in table 1.
- The second-order rate constants can be used to estimate nitroguanidine persistence under specific environmental conditions. In a quiescient water body containing 1 x 10^6 cells/mL and a low nutrient level, the pseudo-first-order rate constant can be estimated to be 3.8 x 10 ^-4/h, from which a half-life can be calculated to be 85 d. However, in a pond containing decomposed organic matter, the microbial population may rise to 1 x 10^7 cells/mL. Assuming 100 ppm organic matter, the pseudo-first-order rate constant is estimated to be 2.3 x 10^-2/h for a half-life of 30 h.
- Under anaerobic conditions, the biotransformation of nitroguanidine was not observed to occur in the absence of glucose and yeast extract. In the presence of these nutrients, the biotransformation proceeded at a much slower rate than observed under aerobic conditions, and therefore was not studied in detail.

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 1 below:

Table 1

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^-4hr^-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^-2 hr^-1, and the half-life will be 30 hours.

Validity criteria fulfilled:

not applicable

Conclusions:

In surface waters with microorganisms adapted to nitroguanidine, the test item is subject to biodegradation. Nitroguanidine biotransforms co-metabolically, the rate is dependent on the concentration of organic nutrients.

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, and the aclimatization of microorganisms.

Biotransformation of nitroguanidine can occur under both aerobic and anaerobic conditions when the microorganisms are adapted sufficiently. The biotransformation of nitroguanidine in surface waters was shown to be a co-metabolic process in which the organisms could degrade nitroguanidine after an aclimatization phase, and only in the presence of other organic nutrients (such as nutrient broth) and nor as a sole carbon and energy source.

Nitroguanidine biotransforms co-metabolically; in the absence of of extra organic nutrients the second-order rate constant was (3.8 +/- 0.9) x 10^-10mL/cell/h and half-life estimates for aerobic, aquatic biotransformation range from 1 to 85 d.

Cyanamide seems to be an end product of nitroguanidine use and no intermediate biotransformation products were observed.

Description of key information

Ultimate degradation in surface waters: Kaplan et al., 1982, incubated solutions of nitroguanidine with activated sludge microorganisms and nutrient broth concentrations ranging from 0.5 to 8.0 g/L under aerobic and anaerobic conditions. The concentration of nitroguanidine was measured (HPLC) over time. Nitroguanidine was not susceptible to aerobic biodegradation in activated sludge, and it was stable under sterile reducing conditions. Nitroguanidine was co-metabolized by anaerobic sludge microorganisms to nitrosoguanidine after acclimation of the microorganisms. Haag et al., 1990 & Spanggord et al., 1987 assessed the biotransformation of nitroguanidine under aerobic conditions 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. Nitroguanidine was added to the bottles from the aqueous stock solution and incubated with or without the addition of nutrient broth and yeast extract. Nitroguanidine was co-metabolically biotransformed by adapted microorganisms at a rate that is dependent on the concentration of organic nutrients. In the absence of extra organic nutrients, the second-order rate constant was (3.8 +/- 0.9) x 10-10mL/cell/h and half-life estimates for aerobic, aquatic biotransformation range from 1 to 100d. Cyanamide seems to be an end product of nitroguanidine use and no intermediate biotransformation products were observed. 

Additional information

Ultimate degradation in surface waters:

Kaplan et al., 1982, incubated solutions of nitroguanidine with activated sludge microorganisms and nutrient broth concentrations ranging from 0.5 to 8.0 g/L under aerobic and anaerobic conditions. The concentration of nitroguanidine was measured (HPLC) over time. In these tests, nitroguanidine was not susceptible to aerobic biodegradation in activated sludge, and it was stable under sterile reducing conditions. Nitroguanidine was co-metabolized by anaerobic sludge microorganisms to nitrosoguanidine after acclimation. There was no further microbial reduction of nitrosoguanidine (no aminoguanidine, hydrazine or urea was detected in culture extracts). Nitrosoguanidine decomposed non-biologically and formed cyanamide, cyanoguanidine, melamine, and guanidine. All products were identified by thin-layer chromatography and mass-spectroscopy.

 

Haag et al., 1990 & Spanggord et al., 1987 assessed the biotransformation of nitroguanidine under aerobic conditions 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. Nitroguanidine was added to the bottles from the aqueous stock solution and incubated with or without the addition of nutrient broth and yeast extract.

In these experiments it could be demonstrated that in surface waters nitroguanidine can be biotransformed at a rate that is dependent on the concentration of organic nutrients. Nitroguanidine biotransforms co-metabolically. These results support the outcome of the experiments previously conducted by Kaplan et al., 1982. In contrary to Kaplan et al., 1982, Haag et al., 1990 & Spanggord et al., 1987 were able to measure degradation of nitroguanidine also under aerobic conditions. In the absence of extra organic nutrients, the second-order rate constant was (3.8 +/- 0.9) x 10-10mL/cell/h and half-life estimates for aerobic, aquatic biotransformation range from 1 to 100d. Cyanamide seems to be an end product of nitroguanidine use and no intermediate biotransformation products were observed.

 

 

Sediment simulation testing:

In accordance with REACH Annex IX, column 2, sediment simulation testing (for substances with a high potential for adsorption to sediment) need not be conducted:

- if the substance is readily biodegradable, or

- if direct and indirect exposure of sediment is unlikely.

Nitroguanidine will not adsorb to soil or sediment organic matter fractions. As such, there is no potential for exposure of sediment, even in the event of a direct release of nitroguanidine to aqueous systems (the nitroguanidine will remain in the aqueous phase). Therefore, no sediment simulation testing is necessary.