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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April 2014 to 4 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Principles of method if other than guideline:
The study design was that desribed in the guideline as a pelagic test rather than a suspended sediment test.
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on source and properties of surface water:
Source of test water: Carsington Water, Millfields, Derbyshire, UK
OS Map Ref of Carsington Water: SK 24813 49995
Water designation: 14-023
Sampling date: 28th April 2014
Surface water type: 300 hectare reservoir which stores water from the river Derwent at Ambergate + small quantities of water draining from surrounding grassland.
Water supplier: Land Research Associates, Derby UK
Pre-treatment of inoculum (water): Samples passed through a 100 µm sieve at the time of sampling.
Water acclimation: acclimated in a CT room to 20 °C overnight before flask filling
Details on inoculum:
Water collection company: Land Research Associates, Derby, UK (non GLP)
Physico-chemical characteristics of water at time of collection:
- Visual quality: Non turbid, colourless
- Temperature: 11.0 °C
- Oxygen: 104.2 % saturation
- Conductivity: 0.14 ppt
- pH 8.32
- 5 day BOD: 1.23 mg O2/L (determined by Chemex, Cambridge UK, non GLP)

Water physico-chemical characteristics certified by: Agvise, Northwood, North Dakota, US (GLP)
- pH - 7.8
- Calcium - 40 ppm
- Magnesium - 5.6 ppm
- Hardness - 123 mg equiv. CaCO3/L
- Conductivity - 0.31 mmhos/cm
- Total Suspended Solids - 8 ppm
- Total Organic Carbon - 2.5 ppm
- Dissolved Organic Carbon- 2.5 ppm
- Ammonical Nitrogen - 0.7 ppm
- Total Nitrogen - 3.2 ppm
- Nitrate (of total Nitrogen) - 0.6 ppm
- Nitrite (of total Nitrogen) - 1.5 ppm
- Total Phosphorous - 0.1 ppm
- Reactive Phosphorous - 0.1 ppm
Duration of test (contact time):
ca. 61 d
Initial conc.:
ca. 10 µg/L
Based on:
test mat.
Initial conc.:
ca. 100 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
test mat. analysis
Details on study design:
- Number of flasks: 48 (24 per dose concentration) dosed with test substance, 8 sterile controls (no inoculum), 4 positive controls (14C benzoic acid), 4 flasks for monitoring test conditions with unlabelled test substance at both 10 µg/L and 100 µg/L concentrations.
- Test material application: high purity water for dosed and inoculated vessels, positive controls and flasks for monitoring test conditions. 50 %
methanol was used to prepare the stock solution for the sterile controls
- Test substance concentrations: 10 and 100 µg/L, verified by LSC and TLC
- Experimental design exposed inoculum: 100 ml borosilicate glass conical flask, attached to incubation system to pass uniform rate moistened air through vessel, connected to a series of three traps (first and second contained 2M potassium hydroxide, third contained 1M hydrochloric acid to trap volatile products)
- Test system temperature: incubated at 20±2 °C, in the dark
- Vessel conditions: aerobic (maintained by stirring and passage of moist air through vessels)
- Sampling frequency dosed vessels: 0, 4, 7, 14, 21, 28, 35, 42, 49, 61 days
- Sampling frequency positive controls: 7, 14 days
- Sampling frequency sterile controls: 61 day
Reference substance:
benzoic acid, sodium salt
Remarks:
Phenyl-U-14C-labelled, purity 98.7 %, specific activity 18.8 MBq/mg
Test performance:
- On day 0 the mean recovery of radioactivity from the test vessels ranged from 100.5 to 101.0 % of the applied radioactivity, indicating the work up method was suitable.
- Measurement of pH and redox potential throughout the test showed the test water remained aerobic and within the environmentally relevant pH range throughout the study.
- The overall mass balance for both of test substance concentrations was 100.9 % and 101.3 % (mean values) of the applied radioactivity for the 10 µg/L and 100 µg/L concentrations, respecitvely.
- The positive control flasks (days 7 and 14) showed acceptable recoveries of 66.8 and 67.6 %, mostly as 14CO2, which demonstrated that the test water
had acceptable levels of biological activity.
Compartment:
other: water, material (mass) balance
% Recovery:
100
Key result
% Degr.:
ca. 0
Parameter:
test mat. analysis
Sampling time:
61 d
Remarks on result:
other: 10 µg/L test concentration
Key result
% Degr.:
ca. 0
Parameter:
test mat. analysis
Sampling time:
61 d
Remarks on result:
other: 100 µg/L test concentration
Key result
% Degr.:
ca. 1.1
Parameter:
CO2 evolution
Sampling time:
61 d
Remarks on result:
other: 10 µg/L test concentration
Key result
% Degr.:
ca. 0.5
Parameter:
CO2 evolution
Sampling time:
61 d
Remarks on result:
other: 100 µg/L test concentration
Key result
Compartment:
natural water: freshwater
DT50:
> 1 000 d
Type:
other: simple first order
Temp.:
20 °C
Remarks on result:
other: 10 µg/L concentration
Key result
Compartment:
natural water: freshwater
DT50:
> 1 000 d
Type:
other: simple first order
Temp.:
20 °C
Remarks on result:
other: 100 mg/L concentration
Transformation products:
yes
No.:
#1
Details on transformation products:
The test substance remained stable over the duration of the test, in all test replicates. TLC and LC-FTMS analysis confirmed parent test substance to be the only component present in the water samples throughout the study. CO2 levels at the end of the study ranged between 0.5 and 1.1 % of the applied radioactivity (100 and 10 µg/L, respectively).
Evaporation of parent compound:
not measured
Volatile metabolites:
yes
Residues:
no
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- None

MINOR TRANSFORMATION PRODUCTS
- CO2: See "mineralisation" below

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
None

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 1.1 % (10 µg/L); 0.5 % (100 µg/L)

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: None

The mean recovery of radioactivity from the test substance treated samples on day zero ranged from 100.5 to 101.0 % of the applied
radioactivity. These results indicate that the work-up method was suitable for the recovery of the applied test substance in the test matrix.
Results with reference substance:
Flasks dosed with non-labelled test substance (reference flasks):
- No systematic difference was found for the monitored parameters (pH, oxygen saturation and redox potential) between the flasks dosed with
10 µg/L and 100 µg/L of test substance.
- The results demonstrate that the test vessels remained aerobic and within the environmenally relevant pH range throughout the study

Flasks dosed with benzoic acid (positive controls)
- Flasks sampled on days 7 and 14.
- Recoveries of ca. 66.8 and 67.6 % (days 7 and 14 respectively) of the applied radioactivity were determined mostly in the form of CO2.
- The percentage of radioactivity in the CO2 represents 82.6 % and 89.9 % of the recovered radioactivity.
- The loss of activity in the positive controls may have been associated with incomplete trapping of [14C]-CO2 or incorporation into biomass.
- The results from the positive control demonstrate that the inoculum (the water) had sufficient biological activity for the study.
Validity criteria fulfilled:
yes
Conclusions:
The test substance was found to be essentially stable in the test system consisting of natural water incubated in the dark at 20 ± 2 °C for 61 days.
Positive controls demonstrated that the test water showed acceptable levels of biological activity and that the work up method was capable of effectively recovering [14C]-CO2 from test replicates.
Executive summary:

The biotransformation of 1-cyanoguandine was studied in filtered pond water (pH 8.32, dissolved organic carbon 2.5 ppm) from the United Kingdom for 61 d under aerobic conditions in the dark at 20 ºC. 1-Cyanoguandine was applied at a rate of 10 and 100 µg/L (as test material). The experiment was conducted in accordance with OECD guideline 309 (13 Apr 2004), and in compliance with the OECD principles of GLP. The test system consisted of borosilicate glass conical flasks with ground glass joints at the neck, attached with traps for the collection of CO2 and basic volatile compounds. Samples were analysed at 0, 4, 7, 14, 21, 28, 35, 42, 49, and 61 days of incubation. The water samples were directly taken from the sample flasks and analysed by TLC. Identification of transformation products was done by TLC and LC-MS, as appropriate.

Material balance was 100.9 % (10 µg/L) and 101.3 % (100 µg/L) of the applied radioactivity. The concentration of parent compound did not significantly decrease from 100 % at day 0 to the end of study period. The half-life/DT50 (50 % decline time) of 1-cyanoguandine in aerobic water was > 1000 days. Accordingly, the parent compound can be considered to be non-biodegradable in filtered surface water. No biotransformation occurred.

At study termination, evolved 14CO2 accounted for 1.1 % (10 µg/L) and 0.5 % (100 µg/L) of the applied radioactivity, respectively. No unidentified radioactivity was reported in the study.

In the sterile controls, the degree of degradation was not different from treatment samples (ca. 100 % of the radioactivity attributable to the parent compound).

Endpoint:
biodegradation in water: sediment simulation testing
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because direct and indirect exposure of sediment is unlikely
other:

Description of key information

The biotransformation of 1-cyanoguandine was studied in filtered pond water over a period of 61 days, according to OECD guideline 309. At study termination, evolved [14C]-CO2 accounted for 0.5-1.1 % of the applied test material,

and the concentration of parent compound was still close to 100 %. The mass balance ranged between 100.9 and 101.3 %. The degradation half life (DT50) in surface water was calculated to be > 1000 days.
1 -Cyanoguandine is not expected to adsorb to sediment (see section 5.4.1, adsorption/desorption). Therefore, there is no potential for exposure of sediment, even in the event of a direct release of 1-cyanoguanidine (dicyandiamide] to aqueous systems. In accordance with REACH Annex IX, column 2, simulation testing of degradation in the sediment is not required.

Key value for chemical safety assessment

Additional information

The biotransformation of 1-cyanoguandine was studied in filtered pond water (pH 8.32, dissolved organic carbon 2.5 ppm) from the United Kingdom for 61 d under aerobic conditions in the dark at 20 ºC. 1-Cyanoguandine was applied at a rate of 10 and 100 µg/L (as test material). The experiment was conducted in accordance with OECD guideline 309 (13 Apr 2004), and in compliance with the OECD principles of GLP. The test system consisted of borosilicate glass conical flasks with ground glass joints at the neck, attached with traps for the collection of CO2 and basic volatile compounds. Samples were analysed at 0, 4, 7, 14, 21, 28, 35, 42, 49, and 61 days of incubation. The water samples were directly taken from the sample flasks and analysed by TLC. Identification of transformation products was done by TLC and LC-MS, as appropriate.

Material balance was 100.9 % (10 µg/L) and 101.3 % (100 µg/L) of the applied radioactivity. The concentration of parent compound did not significantly decrease from 100 % at day 0 to the end of study period. The half-life/DT50 (50 % decline time) of 1-cyanoguandine in aerobic water was > 1000 days. Accordingly, the parent compound can be considered to be non-biodegradable in filtered surface water. No biotransformation occurred.

At study termination, evolved 14CO2 accounted for 1.1 % (10 µg/L) and 0.5 % (100 µg/L) of the applied radioactivity, respectively. No unidentified radioactivity was reported in the study.

In the sterile controls, the degree of degradation was not different from treatment samples (ca. 100 % of the radioactivity attributable to the parent compound).