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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16/08-1988 - 18/09/1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge from oxidation ditch on premises of TNO, Delft, The Netherlands
- Preparation of inoculum for exposure: homogenization of original sludge (4.2 g/L 71% organic matter) of solid substance) and 0.2ml of supernatant used to inoculate 200mL of medium in each flask, inoculum added: 3mg/L organic matter
Duration of test (contact time):
33 d
Initial conc.:
10 mg/L
Based on:
test mat.
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: as described in OECD 301, some adaptation see below
- Additional substrate: NH4Cl
- Test temperature: 20°C
- pH: 7.8 (start of the test)
- pH adjusted: no
- Suspended solids concentration:
- Continuous darkness: yes/no


TEST SYSTEM
- Culturing apparatus: Sapromat flasks provied with a carbon dioxide trap filled with soda lime pellets with indicator (Merck)
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Sapromat containing oxygen supply, CO2 absorbant, magnetic stirrer
- Measuring equipment: automatic system for registration of the oxygen consumption

SAMPLING
- Sampling frequency: oxygen consumption registered continuously for at least 28 days, if degradation was not complete extended to 42 days

CONTROL AND BLANK SYSTEM
- Toxicity control: sodium acetate



Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (DOC removal)
Value:
0
Sampling time:
56 d

The biodegradability of the substance guanidine nitrate was determined by a method derived from the OECD Guideline 301C; Ready Biodegradability: Modified MITI Test, using oxygen consumption as test criterion.

This test was specifically designed in order to study the biodegradability of test compounds with a relatively low carbon content and a relatively high content of oxidizable nitrogen.

The test was carried out using an automatic system (Sapromat) for registration of oxygen consumption and involving a better buffered medium than given in·the Guideline.

In addition to the normal controls on inoculum activity and toxicity, a control on the suitability of the test substance as a nitrogen source was also included. The control tests showed that the activity of the inoculum was sufficient and that no clear effects of the test substance on the inocculum activity (degradation of Sodium Acetate) was found on 10mg/l and 30mg/l.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Guanidinium hydrochloride is not readily biodegradable under this test conditions.
Executive summary:

The ready biodegradation of Guanidine hydrochloride was investigated in a study conducted according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test) over a period of 33 days and using activated sludge as inoculum. The biodegradation rate was determined by measurement of oxygen consumption. Inoculum blank and control with the reference substance sodium acetate were performed. The study is regarded as reliable without restrictions and satisfies the guideline requirements for ready biodegradation.

The test item proved to be not biodegradable under the test conditions.

The functional control reached the pass level >60% after 14 d.

The teset item does not influence the degradation of Sodium acetate at 30mg/l (toxic control).

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Biodegradation Screening and Enrichment Culture Development
Water samples were obtained from two streams in the vicinity of a nitroguanidine pilot production facility
(Hansen and Kill Creeks, Sunflower Army Ammunition Plant. Desoto. KA) and from several stream
(Carroll Creek) and river (Monocacy) sites in the vicinity of Frederick, MD.
Samples were collected aseptically from just beneath water surfaces and chilled during shipment to the laboratory.
Biodegradation studies were initiated within 48 hours of samples collection.
Biodegradation screening was carried out in shake flasks that contained 250 mL of water
samples. 11 mg/L guanidinium (20 mg/L guanidine nitrate) or 17 mg/L arginine. and buffer
(0.2% potassium phosphate. pH 7). Flasks were incubated with agitation at 25° C in the
dark. Aliquots (2 mL) were withdrawn throughout the course of the experiments and assayed
for the test chemicals spectrofluorometrically as described below. Numbers of total bacteria
were determined in triplicate after plating of serial dilutions for 4 days at 25° C on
Standard Methods Agar (SMA. Difco). Hatched controls for the experimental flasks consisted
of water samples that had been autoclaved for 15 min prior to the addition of the test
chemicals.
Aliquots (10 mL) from screening flasks showing guanidinium degradation were used to
inoculate shake flasks containing 20 and 40 mg/L guanidine nitrate in 90 mL of buffered salts
medium from which nitrogen-containing compounds were omitted (BSM-N).16 Following disappearance
of the cation at 250 C, the process was repeated stepwise with 20 to 40 mg/L guanidine
nitrate increments until enrichment cultures were developed; these were serially passed at
300 mg/L guanidine nitrate (168 mg/L guanidinium). Cultures developed in this manner could
be maintained on either guanidine nitrate or hydrochloride with little, if any, apparent
difference in the time required for degradation of the cation. Enrichment microorganisms
grew as clumps finely dispersed throughout the medium that enlarged as guanidinium degradation
progressed; because of their clumped nature, plate counting was variable and ATP
(adenosine triphosphate) measurements were used for biomass estimations.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
surface water
Duration of test (contact time):
22 d
Initial conc.:
20 mg/L
Key result
Parameter:
other: Guanidinium Ion analysis
Value:
100
Sampling time:
20 d

Summary of Guanidinium Degradation in Various Surface Waters

(Degradation rate after days, Bacterial titer in CFU)

Carrol Creek, USA
52 d =   0 %, initial bacterial titer  2.4 x 10E4/ml
68 d = 100 %
Monocacy, USA
20 d =   0 %, initialer Bakterientiter 2.0 x 10E5/ml
28 d = 100 %
Hansen Creek, USA
11 d =   0 %, initialer Bakterientiter 1.3 x 10E5/ml
20 d = 100 %

Guanidinium plus 500 mg/l glucose and arginine were degraded in parallel

samples from all sites in 3 and 2 days, respectively.

Validity criteria fulfilled:
not applicable
Interpretation of results:
inherently biodegradable
Conclusions:
Guanidinium nitrate shows inherent biodegradability in a degradation test with river water. River water was sampled downstream of an amunition plant and contained adapted bacteria.
Executive summary:

Results demonstrate guanidinium-ion degradation in a variety of water samples.The inoculum is expected to be adapted as it is originated downstream an ammunition factory. For this reason, the substance is classified as inherently instead of readily biodegradable.

Degradation half-life is calculated to be 5 days in one case.

Times prior to enhanced microbial activity on guanidinium were variable and tended to be shortest

in samples from sites known to have received nitroguanidine wastewater discharges

(Hansen Creek and Kill Creek). Decreases were not evident for periods of from 11 to 52 days,

after which time guanidinium concentrations decreased linearly, or nearly so, to below detectable

levels in from 6 to 15 days. Both guanidinium in the presence of glucose and arginine were degraded

in 3 and 2 days, respectively, in samples from all sites.

Thus, microorganisms in geographically and temporally different samples are capable of

degrading guanidinium following prolonged periods of incubation; but compared to the amino

acid arginine, which bears a guanidine group, its degradation is extremely slow. Periods

prior to detectable degradation suggest that such microorganisms, although widely distributed,

were initially present in low numbers, even in samples mediating the earliest degradation

of guanidinium (Hansen Creek and Kill Creek). Likewise, microorganisms capable of

degrading guanidinium in the presence of the added carbon source are widely distributed

(also, see below), but the failure to detect degradation for the first several days of

incubation in unamended samples suggests that the process should result in little guanidium

degradation in surface waters.

Description of key information

Guanidine chloride is inherently biodegradable.

This was shown in a non guideline, read-across study with Guanidine nitrate (Mitchell 1987). Test conditions especially test substance concentration and bacteria concentration are comparable to guideline OECD301, inoculum is expected to be adapted.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information

Guanidine chloride is inherently biodegradable.The relevant study is a read-across study, made with Guanidinium nitrate. Read-across is justified as both salts are completely dissociated and for both salts the Guanidinium ion is the component to determine velocity of biodegradation (see detailed justification below).

Mitchell (1987) followed the biodegradation of Guanidinium nitrate in different surface water samples by chemical analysis. No guideline was followed but the test conditions were similar to OECD 301 with the exception that no inocculum but the bacteria naturally occurring in river water were used. The inoculum is expected to be adapted as it is originated downstream an ammunition factory. For this reason, the substance is classified as inherently instead of readily biodegradable.

The test substance concentration (20mg/l) and bacteria concentration (105CFU/ml) were comparable to OECD Guideline 301. Without the addition of a carbon source, only little decrease of Guanidinium could be observed for the first 11 days of incubation. After this time period a degradation rate of approximately 0.07mg/L/hour of Guanidinium was detected and after 20 days Guanidinium was not detectable anymore. The parallel test of a sterilised batch gave no degradation of Guanidinium. In the presence of 500mg/L glucose, after the first day of incubation a significant decrease of Guanidinium could be detected and after 2 to 3 days the level decreased under the detection limit. Degradation half-life is calculated to be 5 days.

According to this reliable study Guanidinium nitrate is inherently biodegradable.

The ready biodegradation of Guanidine hydrochloride was investigated in a study conducted according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test) over a period of 33 days and using activated sludge as inoculum (Alzchem 1990). The biodegradation rate was determined by measurement of oxygen consumption. Inoculum blank and control with the reference substance sodium acetate were performed. The study is regarded as reliable without restrictions and satisfies the guideline requirements for ready biodegradation.

The test item proved to be not biodegradable under the test conditions.

The functional control reached the pass level >60% after 14 d. The test item does not influence the degradation of Sodium acetate at 30mg/l (toxic control).

The biodegradability of Guanidine chloride is supported by positive biodegradation study results in simulation tests in water (Mitchell in Chemosphere (1987)) and soil (Mitchel in Bull. Environ. Contam. Toxicol. (1987).).

As is usual in ecotoxicology for this endpoint, the positve biodegradation screening test with river water superimposes the negative result according to AlzChem (1990). This proposition is supported by the Reach guideline R.7B, May 2008 (version 1.1), R.7.9.4.1 Laboratory data on degradation/biodegradation, BIODEGRADATION, READY BIODEGRADABILITY (page 176): "Realising that ready biodegradability tests may sometime fail because of the stringent test conditions, in general, and the differences among the individual tests in terms of their stringency, consistent positive test results from test(s) should generally supersede negative test results."

Justification for read-across:

Guanidine hydrochloride and guanidine nitrate dissociate in aqueous media to yield the guanidine ion and the respective anion. Therefore it is reasonable to discuss the effects of the ions separately. The chloride ion is a naturally occurring essential ion in human beings with well-known metabolism and mechanisms of action as described in standard textbooks on pharmacology and physiology. As well it is found as salt in the Earth´s crust and is dissolved in seawater. Effects of guanidine hydrochloride are expected to be based primarily on the guanidine ion. The physiological processing of the guanidine ion is expected to be independent of the individual source. Therefore read-across from guanidine nitrate for effects of guanidine dissociated from guanidine hydrochloride is considered valid. This strategy is supported by a quite similar toxicological profile of both substances, as shown in acute toxicity, irritation, sensitization and genotoxic studies.

A more detailed justification for read-across is attached in IUCLID chapter 13.