Sodium chlorate

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

GLP compliance:

yes

Specific details on test material used for the study:

A sample of sodium chlorate was received from Arkema on 2002.11.02
- batch no.: 1E0103WF
- Purity: 99,66%
-water solubility: 715 g/L
-vapour pressure: <3.5 10-5 Pa; 25 C
-log Kow: <-1
-dissociation constant: not relevant
-hydrolysis: stable
-solubility in organic solvents: mixtures of chlorate and organics are explosive

Radiolabelling:

no

Oxygen conditions:

aerobic/anaerobic

Inoculum or test system:

natural sediment

Details on source and properties of sediment:

The sediments used in this studie were sampled from two different locations.
Sediments were sieved trhough screens with openings of 2 mm diameter to improve the uniformity of the substrate application. Nex, the sediments were preconditioned by incubating the sediments for one week at 20+/- 2 C.

One sediment was collected from a stream fed with ground water (Fonteinallee, Heveadorp, the Netherlands).
-low organic content
-sampled at depth of 0-10 cm (aerobic) and 10-20 cm (anaerobic)
-aerobic sediment was grey to brown and had a soil like smell
-anaerobic sediment was grey to brown and had a sulphide smell

The second sediment was taken from the Oostvaardersplassen (OVP) (Knardijk, Lelystad, the Neterlands).
-high organic content
-sampled at depth of 0-5 cm (aerobic) and 15-25 cm (anaerobic)
-aerobic sediment was grey to black and had a slightly sulphide smell
-anaerobic sediment was black and had a sulphide smell


FOR MORE CHARACTERISTICS OF THE SEDIMENTS AND OVERLYING WATER SEE TABLE I AND II IN THE ATTACHED REPORT

Duration of test (contact time):

>= 28 - <= 56 d

Initial conc.:

25 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

Test procedures
The test was performed according to an OECD test guideline (proposal for a new guideline 308). The biodegradation of sodium chlorate was evaluated in two sediments.
The sediments were preincubated at 20 ± 2°C for one week (from start (day -7) to the introduction of chlorate (day 0). After this week chlorate was introduced at a
concentration of 25 mg/L in the water phase. The initial concentration of chlorate in the test was calculated on the baSis of the maximum application rate of 250 kg active
substance per hectare, the internal diameter of the test flask of 8-cm, and a water column depth of ±13.5 cm (deviation from the study plan). The sediment layer was ±4.5 cm. The
test substance was dissolved in an appropriate amount of deionized water allowing addition of 0.2 mL of the stock solution to the aqueous phase of the water sediment system. For
each sediment, there was one treatment i.e. test substance added to the water sediment system. The test was carried out in bottles closed with butyl septa (anaerobic incubations)
or parafilm (aerobic incubations). During the test it was noted that the oxygen concentration in the overlying water decreased in both aerobic treatments. To prevent
depletion of oxygen in the aerobic incubations, the overlying water was aerated at such a rate that the overlying water was oxygen saturated throughout the duration of the test
(deviation of study plan). Anaerobic conditions were established at the start of the test by flushing the gas phase with nitrogen gas. Degradation of chlorate in the sediments (aerobic
and anaerobic) was followed in duplicate bottles. The septa and parafilm ensured that the water level was maintained for the duration of the test. Sampling in duplicate bottles was
performed with minimal disturbance of the sediment and the overlying water using a glass pipe with a diameter of ± 10 mm.
The test flasks were incubated at 20 ± 2°C. The experiments with sediment from OVP and Heveadorp were run for 28 and 56 days, respectively. Samples of the overlying water
and the sediment were withdrawn for analyses at day 0, 3, 7, 10, 14, 28, 42 (both sediments), and 56 (only Heveadorp). The following time intervals were adopted for the
anaerobic bottles starting after introducing the anaerobic conditions; day 0, 1, 3, 7, 10, 14, 21, 28 (both sediments), and 56 (only Heveadorp).
Two controls (bottles without chlorate addition) of both Heveadorp and OVP were incubated to determine the microbial biomass at the end of the test. These control bottles
were also used to measure the formation of chloride in the absence of chlorate. This was decided after the observation of excess chloride formation. Data on chloride formation in
the absence of chlorate enabled a better assessment of the chlorine mass balance.


Evaluation of data
The loss of parent compound data could not be fitted to various equations described by Alexander (1984). Regression analyses were therefore determined with a number of data
pOints (usually disregarding the first measurements) using SlideWriteplus@ (version 4.0) software of Advanced Graphics Software Inc. The initial chlorate concentration was
calculated by dividing the total amount of sodium chlorate added with the total volume of water (overlying water and the water in the sediment phase). DTso, DT7s, and OT9o, were
determined by plotting the respective times required to achieve 50, 75 and 90% reduction of the calculated initial sodium chlorate concentration.

Test performance:

Test conditions
The sediment systems were incubated at 20 ± 2°C. The pH values in the sediments and overlying waters ranged from 6.1 to 7.3. The redox potential of the sediments (aerobic
and anaerobic incubations) varied from -76 to -180 mV. The redox potential of OVP sediment was slightly lower than Heveadorp sediment. The redox potential in the
overlying water of the aerobic incubations was always higher than 240 mV. This high redox potential is in agreement with the oxygen concentrations found. The redox
potential in the overlying water of the anaerobic incubations decreased during the incubation. The decrease of the redox potential during the test period is probably a
reflection of the degradation of chlorate. No or minor concentrations of oxygen were detected in the sediments. High concentrations of oxygen were detected only in the
overlying water of the aerobic incubations. Finally, the organic carbon contents in the overlying water decreased from 54-64 mg/L to 26-32 mg/L (Tables I and II report).

Compartment:

other: aerobic OVP sediment

% Recovery:

122

Remarks on result:

other: almost all chlorate- chlorine recovered as chloride at the end of the test

Compartment:

other: aerobic heveadorp sediment

% Recovery:

90

Compartment:

other: anaerobic heveadorp sediment

% Recovery:

88

Compartment:

other: anaerobic OVP sediment

% Recovery:

92

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

aerobic OVP sediment

Sampling time:

3 d

Remarks on result:

other: 90% biodegradation achieved in < 3 days

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

OVP aerobic water

Sampling time:

12 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

OVP anaerobic sediment

Sampling time:

1 d

Remarks on result:

other: 90% biodegradation achieved in <1 day

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

OVP anaerobic water

Sampling time:

15 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

Heveadorp aerobic sediment

Sampling time:

34 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

heveadorp aerobic water

Sampling time:

42 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

heveadorp anaerobic sediment

Sampling time:

51 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

heveadorp anaerobic water

Sampling time:

54 d

Compartment:

water

DT50:

8 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: aerobic OVP water

Compartment:

sediment

DT50:

< 3 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: aerobic OVP sediment

Compartment:

water

DT50:

9 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: anaerobic OVP water

Compartment:

sediment

DT50:

< 1 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: anaerobic OVP sediment

Compartment:

water

DT50:

20 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: aerobic heveadorp water

Compartment:

sediment

DT50:

18 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: aerobic heveadorp sediment

Compartment:

water

DT50:

29 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: anaerobic heveadorp water

Compartment:

sediment

DT50:

24 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: anaerobic heveadorp sediment

Transformation products:

yes

Remarks:

mass balances of chlorine in the water and sediment systems strongly indicate that chlorate is converted into chloride.

No.:

#1

Evaporation of parent compound:

not measured

Remarks:

not expected based on phys chemical properties of chlorate and the obtained chlorine mass balance in the water sediment system

Volatile metabolites:

no

Residues:

no

Details on results:

The initial concentration of chlorate in the overlying water was calculated by assuming an even distribution of the test substance in the overlying water and the water in the
sediment. Sodium chlorate was applied at a concentration of 25 mg/L (234 uM) in theoverlying water. The volume of the water in the sediment was extracted from the dry
weight determinations. Due to the immediate start of chlorate reduction and limited mixing of the overlying water at the start of the test it is not expected that the chlorate
concentrations measured at the start agree with the calculated initial chlorate concentration (Table III, and IV, Annex B). The results of the degradation of sodium
chlorate are given in Tables III and IV and Annex E. The results of Tables III and IV are also graphically presented in Figures 1-8. It was not possible to fit the depletion curves
with equations described by Alexander (1994). The times required for 50, 75 and 90% degradation of chlorate (DT50, DT75, and DT90) in the sediments and overlying water were
therefore determined by plotting (Figures 1-8). For this purpose the calculated initial concentrations of the water sediment systems were used (Annex B). These initial
concentrations were multiplied by 0.5, 0.25 and 0.1 to obtain DT50, DT75, and DT90 values, respectively. The DT50 values of Heveadorp ranged from 18 to 29 days. The DT50 values of OVP are low compared to Heveadorp probably reflecting the different organic carbon contents of both sediments. Chlorate concentrations in the sediments of OVP were low from the start of the test resulting in a very low DT50 value.

Mass balances of chlorine in water sediment systems may be used to demonstrate complete conversion of chlorate into chloride. Excess of chloride was produced in the
water sediment systems. This excess already strongly indicates that the chlorate added was reduced to chloride. The controls incubated for the determination of the biomass
concentration enabled the assessment of the "endogenous" formation of chloride in the sediment water systems (Table VI). The chloride formed in the control water sediment
systems was subtracted from the chloride produced in the sediment water systems with chlorate. The chloride concentrations calculated ranged from 181 to 298 !!mol/L (Table VI)
This demonstrates that almost 100% of the chlorate-chlorine in the aerobic OVP sediment water system is recovered as chloride. More than 100% is found in the other sediment
water systems. The mass balances demonstrate that chlorate is completely reduced to chloride. Moreover, the degradation pathway of chlorate proving the complete conversion
of chlorate into chloride has been described comprehensively in the open literature.

Validity of the test

At day 0 the chlorate concentrations in the sediment water systems of OVP (aerobic), OVP (anaerobic)1 Heveadorp (aerobic) and Heveadorp (anaerobic) were 241, 175, 174, and 173 uM, respectively. These concentrations are calculated with the chlorate concentrations

measured at day 0 (Tables III and IV) and the water in the systems (Annex B) by assuming a uniform distribution and no degradation. The calculated initial chlorate concentrations in the sediment water systems of OVP (aerobic), OVP (anaerobic), Heveadorp (aerobic) and Heveadorp (anaerobic) were 1971 1951 1971 and 188 uM respectively (Annex B). The recoveries in the sediment water systems of OVP (aerobic), OVP (anaerobic) Heveadorp (aerobic) and Heveadorp (anaerobic) were therefore 122, 90, 88, and 92%, respectively. These recoveries are in the prescribed range of 70 to 110% with the exception of OVP (aerobic). This exception is probably caused by the uneven distribution of chlorate at the start of the test. The chloride formed in the sediment water systems of OVP (aerobic), OVP (anaerobic), Heveadorp (aerobic) and Heveadorp (anaerobic) was 181, 242, 298, and 262 uM, respectively (Table VI). This strongly indicates that almost all chlorate-chlorine was recovered as chloride at the end of the test.

The limit of detection of the analytical method for the test substance was calculated with areas measured at 0.31 mg/L sodium chlorate (lowest concentration used for the calibration curves) given in Table VII. The average value of the areas is 33884 (0.31 mg/L) with a standard deviation of 1612 (0.15 mg/L). The limit of detection (LOD) is calculated by multiplying the 0.15 with 3. The LOD i.e. 0.45 mg/L is therefore 1.8% of the applied dose (minor deviation from the required 1% in the OECD Guideline). The limit of quatification (LOQ) is 0.9 mg/L. This was calculated by multiplying the standard deviation by a factor of 6.3. Standard deviations of the triplicate chlorate analyses of less than 1 % demonstrate the repeatability of the analysis. First order kinetics were not applied (curves were more complex) to calculate half-lives. DT50, DT75, and DT90 values are used to quantify the degradation rates.

Validity criteria fulfilled:

yes

Remarks:

see "validity of the test" reported above

Conclusions:

The biodegradation of chlorate in aerobic and anaerobic sediments was assessed according the OECD guidelines for testing of chemicals (OECD 308) in compliance with the OECD principles of GLP. The validity criteria described in the OECD guideline were met with the minor deviation of the LOD which was 1.8% of the applied dose instead of 1%.

Chlorate was reduced at higher rates in sediments with a high organic carbon content compared to sediments with a low carbon content. The DT50 for sediment with high organic carbon content was 8 days in water phase and less than 3 days in sediment under aerobic conditions and 9 days in water phase and less than 1 day in sediment under anaerobic conditions. The DT50 for sediment with low organic carbon content was 20 days in water phase and 18 days in sediment under aerobic conditions and 29 dasy in water phase and 24 days in sediment under anaerobic conditions.

Executive summary:

The aerobic and anaerobic transformation of chlorate in two sediments were assessed according to OECD guidelines for testing of chemicals (proposal for a new guideline 308), and in compliance with the OECD principles of Good Laboratory Practice. A sediment with a high organic carbon content (Oostvaardersplassen) and a sediment with a low organic carbon content (Heveadorp) were used. The transformation rates of chlorate in these sediments and their overlying water under both aerobic and anaerobic conditions were determined by measuring the chlorate depletion. Lag periods were absent or very short in both sediment water systems.

The DT50 , DT75, and DT90 (times required for 50, 75 and 90% degradation of chlorate) in the sediments and overlying water were as follows;

Oostvaarderplassen (OVP); high organic carbon content
	Aerobic Anaerobic
	water	sediment	water	sediment
DT50(days)	8	<3	9	<1
DT75(days)	10	<3	12	<1
DT90(days)	12	<3	15	<1
Heveadorp; low organic carbon content
	water	sediment	water	sediment
DT50(days)	20	18	29	24
DT75(days)	31	26	41	36
DT90(days)	42	34	54	51

Mass balances of chlorine in the water sediment systems strongly indicate that chlorate is converted into chloride. The limit of detection of the analytical method used for chlorate was <2% of the initial amount applied to the system. The repeatability of the analytical method was checked by duplicate analysis.

Reference 2

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

GLP compliance:

yes

Radiolabelling:

no

Oxygen conditions:

aerobic/anaerobic

Inoculum or test system:

natural sediment

Details on source and properties of sediment:

The sediments used in this studie were sampled from two different locations.
Sediments were sieved trhough screens with openings of 2 mm diameter to improve the uniformity of the substrate application. Nex, the sediments were preconditioned by incubating the sediments for one week at 20+/- 2 C.

One sediment was collected from a stream fed with ground water (Fonteinallee, Heveadorp, the Netherlands).
-low organic content
-sampled at depth of 0-10 cm (aerobic) and 10-20 cm (anaerobic)
-aerobic sediment was grey to brown and had a soil like smell
-anaerobic sediment was grey to brown and had a sulphide smell

The second sediment was taken from the Oostvaardersplassen (OVP) (Knardijk, Lelystad, the Neterlands).
-high organic content
-sampled at depth of 0-5 cm (aerobic) and 15-25 cm (anaerobic)
-aerobic sediment was grey to black and had a slightly sulphide smell
-anaerobic sediment was black and had a sulphide smell


FOR MORE CHARACTERISTICS OF THE SEDIMENTS AND OVERLYING WATER SEE TABLE I AND II IN THE ATTACHED REPORT

Duration of test (contact time):

>= 28 - <= 56 d

Initial conc.:

25 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

Test procedures
The test was performed according to an OECD test guideline (proposal for a new guideline 308). The biodegradation of sodium chlorate was evaluated in two sediments.
The sediments were preincubated at 20 ± 2°C for one week (from start (day -7) to the introduction of chlorate (day 0). After this week chlorate was introduced at a
concentration of 25 mg/L in the water phase. The initial concentration of chlorate in the test was calculated on the baSis of the maximum application rate of 250 kg active
substance per hectare, the internal diameter of the test flask of 8-cm, and a water column depth of ±13.5 cm (deviation from the study plan). The sediment layer was ±4.5 cm. The
test substance was dissolved in an appropriate amount of deionized water allowing addition of 0.2 mL of the stock solution to the aqueous phase of the water sediment system. For
each sediment, there was one treatment i.e. test substance added to the water sediment system. The test was carried out in bottles closed with butyl septa (anaerobic incubations)
or parafilm (aerobic incubations). During the test it was noted that the oxygen concentration in the overlying water decreased in both aerobic treatments. To prevent
depletion of oxygen in the aerobic incubations, the overlying water was aerated at such a rate that the overlying water was oxygen saturated throughout the duration of the test
(deviation of study plan). Anaerobic conditions were established at the start of the test by flushing the gas phase with nitrogen gas. Degradation of chlorate in the sediments (aerobic
and anaerobic) was followed in duplicate bottles. The septa and parafilm ensured that the water level was maintained for the duration of the test. Sampling in duplicate bottles was
performed with minimal disturbance of the sediment and the overlying water using a glass pipe with a diameter of ± 10 mm.
The test flasks were incubated at 20 ± 2°C. The experiments with sediment from OVP and Heveadorp were run for 28 and 56 days, respectively. Samples of the overlying water
and the sediment were withdrawn for analyses at day 0, 3, 7, 10, 14, 28, 42 (both sediments), and 56 (only Heveadorp). The following time intervals were adopted for the
anaerobic bottles starting after introducing the anaerobic conditions; day 0, 1, 3, 7, 10, 14, 21, 28 (both sediments), and 56 (only Heveadorp).
Two controls (bottles without chlorate addition) of both Heveadorp and OVP were incubated to determine the microbial biomass at the end of the test. These control bottles
were also used to measure the formation of chloride in the absence of chlorate. This was decided after the observation of excess chloride formation. Data on chloride formation in
the absence of chlorate enabled a better assessment of the chlorine mass balance.


Evaluation of data
The loss of parent compound data could not be fitted to various equations described by Alexander (1984). Regression analyses were therefore determined with a number of data
pOints (usually disregarding the first measurements) using SlideWriteplus@ (version 4.0) software of Advanced Graphics Software Inc. The initial chlorate concentration was
calculated by dividing the total amount of sodium chlorate added with the total volume of water (overlying water and the water in the sediment phase). DTso, DT7s, and OT9o, were
determined by plotting the respective times required to achieve 50, 75 and 90% reduction of the calculated initial sodium chlorate concentration.

Test performance:

Test conditions
The sediment systems were incubated at 20 ± 2°C. The pH values in the sediments and overlying waters ranged from 6.1 to 7.3. The redox potential of the sediments (aerobic
and anaerobic incubations) varied from -76 to -180 mV. The redox potential of OVP sediment was slightly lower than Heveadorp sediment. The redox potential in the
overlying water of the aerobic incubations was always higher than 240 mV. This high redox potential is in agreement with the oxygen concentrations found. The redox
potential in the overlying water of the anaerobic incubations decreased during the incubation. The decrease of the redox potential during the test period is probably a
reflection of the degradation of chlorate. No or minor concentrations of oxygen were detected in the sediments. High concentrations of oxygen were detected only in the
overlying water of the aerobic incubations. Finally, the organic carbon contents in the overlying water decreased from 54-64 mg/L to 26-32 mg/L (Tables I and II report).

Compartment:

other: aerobic OVP sediment

% Recovery:

122

Remarks on result:

other: almost all chlorate- chlorine recovered as chloride at the end of the test

Compartment:

other: aerobic heveadorp sediment

% Recovery:

90

Compartment:

other: anaerobic heveadorp sediment

% Recovery:

88

Compartment:

other: anaerobic OVP sediment

% Recovery:

92

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

aerobic OVP sediment

Sampling time:

3 d

Remarks on result:

other: 90% biodegradation achieved in < 3 days

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

OVP aerobic water

Sampling time:

12 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

OVP anaerobic sediment

Sampling time:

1 d

Remarks on result:

other: 90% biodegradation achieved in <1 day

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

OVP anaerobic water

Sampling time:

15 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

Heveadorp aerobic sediment

Sampling time:

34 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

heveadorp aerobic water

Sampling time:

42 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

heveadorp anaerobic sediment

Sampling time:

51 d

% Degr.:

90

Parameter:

test mat. analysis

Remarks:

heveadorp anaerobic water

Sampling time:

54 d

Compartment:

water

DT50:

8 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: aerobic OVP water

Compartment:

sediment

DT50:

< 3 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: aerobic OVP sediment

Compartment:

water

DT50:

9 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: anaerobic OVP water

Compartment:

sediment

DT50:

< 1 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: anaerobic OVP sediment

Compartment:

water

DT50:

20 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: aerobic heveadorp water

Compartment:

sediment

DT50:

18 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: aerobic heveadorp sediment

Compartment:

water

DT50:

29 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: anaerobic heveadorp water

Compartment:

sediment

DT50:

24 d

Type:

not specified

Temp.:

20 °C

Remarks on result:

other: anaerobic heveadorp sediment

Transformation products:

yes

Remarks:

mass balances of chlorine in the water and sediment systems strongly indicate that chlorate is converted into chloride.

No.:

#1

Evaporation of parent compound:

not measured

Remarks:

not expected based on phys chemical properties of chlorate and the obtained chlorine mass balance in the water sediment system

Volatile metabolites:

no

Residues:

no

Details on results:

The initial concentration of chlorate in the overlying water was calculated by assuming an even distribution of the test substance in the overlying water and the water in the
sediment. Sodium chlorate was applied at a concentration of 25 mg/L (234 uM) in theoverlying water. The volume of the water in the sediment was extracted from the dry
weight determinations. Due to the immediate start of chlorate reduction and limited mixing of the overlying water at the start of the test it is not expected that the chlorate
concentrations measured at the start agree with the calculated initial chlorate concentration (Table III, and IV, Annex B). The results of the degradation of sodium
chlorate are given in Tables III and IV and Annex E. The results of Tables III and IV are also graphically presented in Figures 1-8. It was not possible to fit the depletion curves
with equations described by Alexander (1994). The times required for 50, 75 and 90% degradation of chlorate (DT50, DT75, and DT90) in the sediments and overlying water were
therefore determined by plotting (Figures 1-8). For this purpose the calculated initial concentrations of the water sediment systems were used (Annex B). These initial
concentrations were multiplied by 0.5, 0.25 and 0.1 to obtain DT50, DT75, and DT90 values, respectively. The DT50 values of Heveadorp ranged from 18 to 29 days. The DT50 values of OVP are low compared to Heveadorp probably reflecting the different organic carbon contents of both sediments. Chlorate concentrations in the sediments of OVP were low from the start of the test resulting in a very low DT50 value.

Mass balances of chlorine in water sediment systems may be used to demonstrate complete conversion of chlorate into chloride. Excess of chloride was produced in the
water sediment systems. This excess already strongly indicates that the chlorate added was reduced to chloride. The controls incubated for the determination of the biomass
concentration enabled the assessment of the "endogenous" formation of chloride in the sediment water systems (Table VI). The chloride formed in the control water sediment
systems was subtracted from the chloride produced in the sediment water systems with chlorate. The chloride concentrations calculated ranged from 181 to 298 !!mol/L (Table VI)
This demonstrates that almost 100% of the chlorate-chlorine in the aerobic OVP sediment water system is recovered as chloride. More than 100% is found in the other sediment
water systems. The mass balances demonstrate that chlorate is completely reduced to chloride. Moreover, the degradation pathway of chlorate proving the complete conversion
of chlorate into chloride has been described comprehensively in the open literature.

Validity of the test

At day 0 the chlorate concentrations in the sediment water systems of OVP (aerobic), OVP (anaerobic)1 Heveadorp (aerobic) and Heveadorp (anaerobic) were 241, 175, 174, and 173 uM, respectively. These concentrations are calculated with the chlorate concentrations

measured at day 0 (Tables III and IV) and the water in the systems (Annex B) by assuming a uniform distribution and no degradation. The calculated initial chlorate concentrations in the sediment water systems of OVP (aerobic), OVP (anaerobic), Heveadorp (aerobic) and Heveadorp (anaerobic) were 1971 1951 1971 and 188 uM respectively (Annex B). The recoveries in the sediment water systems of OVP (aerobic), OVP (anaerobic) Heveadorp (aerobic) and Heveadorp (anaerobic) were therefore 122, 90, 88, and 92%, respectively. These recoveries are in the prescribed range of 70 to 110% with the exception of OVP (aerobic). This exception is probably caused by the uneven distribution of chlorate at the start of the test. The chloride formed in the sediment water systems of OVP (aerobic), OVP (anaerobic), Heveadorp (aerobic) and Heveadorp (anaerobic) was 181, 242, 298, and 262 uM, respectively (Table VI). This strongly indicates that almost all chlorate-chlorine was recovered as chloride at the end of the test.

The limit of detection of the analytical method for the test substance was calculated with areas measured at 0.31 mg/L sodium chlorate (lowest concentration used for the calibration curves) given in Table VII. The average value of the areas is 33884 (0.31 mg/L) with a standard deviation of 1612 (0.15 mg/L). The limit of detection (LOD) is calculated by multiplying the 0.15 with 3. The LOD i.e. 0.45 mg/L is therefore 1.8% of the applied dose (minor deviation from the required 1% in the OECD Guideline). The limit of quatification (LOQ) is 0.9 mg/L. This was calculated by multiplying the standard deviation by a factor of 6.3. Standard deviations of the triplicate chlorate analyses of less than 1 % demonstrate the repeatability of the analysis. First order kinetics were not applied (curves were more complex) to calculate half-lives. DT50, DT75, and DT90 values are used to quantify the degradation rates.

Validity criteria fulfilled:

yes

Remarks:

see "validity of the test" reported above

Conclusions:

The biodegradation of chlorate in aerobic and anaerobic sediments was assessed according the OECD guidelines for testing of chemicals (OECD 308) in compliance with the OECD principles of GLP. The validity criteria described in the OECD guideline were met with the minor deviation of the LOD which was 1.8% of the applied dose instead of 1%.

Chlorate was reduced at higher rates in sediments with a high organic carbon content compared to sediments with a low carbon content. The DT50 for sediment with high organic carbon content was 8 days in water phase and less than 3 days in sediment under aerobic conditions and 9 days in water phase and less than 1 day in sediment under anaerobic conditions. The DT50 for sediment with low organic carbon content was 20 days in water phase and 18 days in sediment under aerobic conditions and 29 dasy in water phase and 24 days in sediment under anaerobic conditions.

Executive summary:

The aerobic and anaerobic transformation of chlorate in two sediments were assessed according to OECD guidelines for testing of chemicals (proposal for a new guideline 308), and in compliance with the OECD principles of Good Laboratory Practice. A sediment with a high organic carbon content (Oostvaardersplassen) and a sediment with a low organic carbon content (Heveadorp) were used. The transformation rates of chlorate in these sediments and their overlying water under both aerobic and anaerobic conditions were determined by measuring the chlorate depletion. Lag periods were absent or very short in both sediment water systems.

The DT50 , DT75, and DT90 (times required for 50, 75 and 90% degradation of chlorate) in the sediments and overlying water were as follows;

Oostvaarderplassen (OVP); high organic carbon content
	Aerobic Anaerobic
	water	sediment	water	sediment
DT50(days)	8	<3	9	<1
DT75(days)	10	<3	12	<1
DT90(days)	12	<3	15	<1
Heveadorp; low organic carbon content
	water	sediment	water	sediment
DT50(days)	20	18	29	24
DT75(days)	31	26	41	36
DT90(days)	42	34	54	51

Mass balances of chlorine in the water sediment systems strongly indicate that chlorate is converted into chloride. The limit of detection of the analytical method used for chlorate was <2% of the initial amount applied to the system. The repeatability of the analytical method was checked by duplicate analysis.

Additional information

Assimilatory nitrate reductases

Assimilationis the conversion nitrate into ammonium for anabolic reactions. Nitrate is reduced for this purpose by enzymes to nitrite (assimilatory nitrate reductases), and then to ammonia. The assimilatory nitrate reductases are molybdenum-containing enzymes, which are widespread in bacteria, fungi, yeasts, and algae (Campbell, 2001; Inokuchi et al, 2002; Joseph-Horne et al, 2001; Siverio, 2002). Nitrate and chlorate are structurally analogous to each other and may potentially be incorporated into the same enzyme active site, as is evidenced by various assimilatory nitrate reductasesof micro-organisms and plants. Chlorate reduction by assimilatory nitrate reductases has been detected in bacteria (Escherichia coli) using a mutant (Motohara et al, 1976). Balch (1987) experimented with³⁶Cl chlorate as a tracer to study nitrate uptake. In Skeletonema costatum and Nitzschia closterium chlorate was transported into the cells. The ability to reduce chlorate in whole cells has been shown in Ankistrodesmus braunii and Chlorella fusca both algae (Rigano, 1970; Tromballa and Broda, 1971). Chlorate is also a substrate for assimilatory nitrate reductase of Chloralla vulgaris (Solomonson and Vennesland, 1972). The assimilatory nitrate reductases convert chlorate to a toxic product chlorite. These results demonstrate that there is a potential for chlorate reduction under aerobic conditions provided that organisms are present capable of utilizing nitrate as nitrogen source.

Dissimilatory nitrate reductases

Denitrification is a process by which bacteria convert nitrate to dinitrogen that is lost to the atmosphere. Denitrifying bacteria use nitrate instead of oxygen in the metabolic processes. Denitrification takes primarily place where oxygen is depleted and where there is ample organic matter to provide energy for bacteria. Two types of dissimilatory nitrate reductases have been found. One of them is coupled to a complete denitrifying pathway (membrane-bound nitrate reductases; nitrate reductase A), and the other is a periplasmic protein whose physiological role seems to be the dissipation of excess reducing power. Periplasmic nitrate reductases, responsible for denitrification under aerobic conditions are specific for nitrate and not capable of reducing chlorate (Berks et al, 1994; McEwan et al, 1987). Chlorate reduction in denitrifying bacteria is primarily due to membrane-bound nitrate reductase (nitrate reductase A) activity (Iobbi et al, 1987; Morpeth and Boxer, 1985). The reduction of nitrate and chlorate in cell-free extracts of nitrate-grown Bacillus cereus was investigated by Hackenthal (1965). Chlorate reduction rates in cell-free extract were approximately twice as high as the nitrate reduction rates. De Groot and Stouthamer (1969) found that Proteus mirabilis formed different reductases including a chlorate reductase (chlorate reductase C). Chlorate reductase purified from Proteus mirabilis could only use chlorate as a substrate (Oltman et al, 1976). It was found that chlorate reductase was produced constitutively while nitrate reductases were produced inductively. However, the chlorate reduction in cell-free extracts of nitrate-grown bacteria is primarily due to membrane-bound nitrate reductases (de Groot and Stouthamer, 1969).

Chlorite is produced from chlorate by denitrifying microorganisms (Quastel et al, 1925; Karki and Kaiser, 1979). It was found that the absorption spectrum of dissimilatory nitrate reductase obtained from Escherichia coli after oxidation by chlorate was different from that of normal oxidized cytochrome. It was assumed that this was due to the oxidative deformation of the haem by the reduction product chlorite (Itagaki et al, 1963). Chlorite formed by denitrifying bacteria is degraded through chemical reactions with reducing agents such as the protein of nitrate reductase. In conclusion, chlorate reduction associated with nitrate-respiring organisms is a cometabolic process. The rate of chlorate reduction by denitrifying bacteria is therefore directly linked to the rate of denitrification.

Growth linked biodegradation (anaerobic)

It is now well-known that bacteria have evolved that can grow by the anaerobic reductive dissimilation of chlorate into innocuous chloride. Bacteria capable of growing with chlorate as electron acceptor are widely spread nature. This has been shown by (per)chlorate reduction with various energy substrates with a number of enrichment cultures (Bryan and Rohlich 1954; van Ginkel et al, 1995; Logan, 1998). The ubiquity of (per)chlorate reducing microorganisms was also shown quantitatively by enumerating the (per)chlorate-reducing bacteria in very diverse environments, including soils, aquatic sediments, sludges, and lagoons. In all of the environments tested, the acetate-oxidizing (per)chlorate reducing bacteria represented a significant population, whose size ranged from 2.3 × 10³to 2.4 × 10⁶cells per g of sample (Coates et al, 1999; Wu et al, 2001). Existence of (per)chlorate respiring bacteria have also been demonstrated in marine waters (Logan et al, 2000)

(Per)chlorate reducing microorganisms are easily enriched and isolated from many environments. All of these organisms could grow anaerobically by coupling complete oxidation of reducing agents to reduction of chlorate at high rates (Table). Under fully aerobic conditions, chlorate is not reduced by capable bacteria. Nitrate can also interfere with chlorate reduction (Chaudhuri et al 2002).

 

Table Growth ratesof various cultures capable of reducing chlorate.

Culture	Reducing agent	Rate (h^-1)	Reference
Azospira oryzoa GR-1	Acetate	0.1	Rikken et al (1996)
Dechloromonas Agita sp CKB		0.28	Bruce et al (1999)
Azospira sp KJ	Acetate	0.26	Logan et al (2001)
Dechlorosomonas sp PDX	Acetate	0.21	Logan et al (2001)
Dechlorosomonas sp PDX	Lactate	0.15	Logan et al (2001)
PDA	Acetate	0.18	Logan et al (2001)
PDB	Actate	0.21	Logan et al (2001)
Mixed culture	Acetate	0.56	Logan et al (1998)
Mixed culture	glucose glutamate	0.12	Logan et al (1998)
Mixed culture	Phenol	0.04	Logan et al (1998)

 

Azospira oryzae strain GR-1 (DSM 11199) isolated from activated sludge was the first bacterium studied in more detail (Rikken et al, 1996; Wolterink et al, 2005). When strain GR-1 was grown on acetate, the release of chloride was proportional to the disappearance of chlorate, showing that this compound was completely reduced. The oxidation of acetate is coupled to the reduction of chlorate, whereas chlorite reduction is not affected by the addition of acetate. Azospira oryzae strain GR-1 disproportionates chlorite into molecular oxygen and chloride. For chlorate reduction by Azospira oryzae strain GR-1 the following biodegradation pathway was formulated: ClO₃^- → ClO₂^-→ Cl^-+ O₂. The rapid dismutation of chlorite into chloride and molecular oxygen is the key reaction in the reduction of chlorate. All (per)chlorate-reducing bacteria isolated to date have the ability to dismutate chlorite (Coates and Achenbach, 2004). Complete reduction of chlorate into chloride and molecular oxygen is catalysed by two enzymes. Chlorate is reduced to chlorite by (per)chlorate reductase (EC 1.97.1.1) (Kengen et al, 1999). Chlorate respiration at high rates is made possible by the action of the second enzyme which reduces the toxic chlorite to chloride while producing molecular oxygen. This is mediated by chlorite dismutase (EC 1.13.11.49) (van Ginkel et al 1996; Stenklo et al 2001). Chlorite has never been found to accumulate in solution during bacterial respiration of (per)chlorate.

Aqueous fresh systems

Although sodium chlorate is considered readily biodegradable, the TGD default value does probably not represent the half-live in aerobic surface water. The fate of sodium chlorate in aerobic surface waters is linked to nitrate assimilation rates (cometabolic process).It is therefore important to know the nitrate assimilation rates in any aqueous ecosystems for assessment of the potential of chlorate reduction.An estimate of the nitrate uptake rate is therefore based on data found in the open literature.Compared to nitrate-nitrogen uptake rates, chlorate reduction rates are assumed to be 10 times lower (expert opinion). The total amount of chlorate in the environment compared to nitrate assimilated is probably negligible. Chlorate can therefore be completely transformed. Nitrate-nitrogen uptake rates by phytoplankton in aranged from 0.006 to 0.036µM h-1 (Rojo et al 2008). The nitrate-nitrogen uptake rates by heterotrophic bacteria in the riverrange from 0.001 to 1.44 µM h-1 (Middelburg and Nieuwenhuizen, 2000). Using an environmental chlorate concentration of 0.036 µM – 0.52 µM (Versteegh-Neele-Cleven,1993) half lives ranging from 0.2 day to 18 days can be calculated with the nitrogen uptake rates for phytoplankton (zero order kinetics).

Freshwater with sediment

Reduction of chlorate was found in a water-sediment system under both aerobic and anaerobic conditions (OECD TG 308). Sediments with a high (13% for aerobic sediment and 4.4% for anaerobic sediment) and a low (0.4% both aerobic and anaerobic) organic carbon contents were used. Chlorate was reduced at higher rates in sediments with a high organic carbon content compared to sediments with a low carbon content. This result is consistent with the literature on microbial chlorate reduction. The DT50 for sediment with high organic carbon content was 8 days in water phase and less than 3 days in sediment under aerobic conditions and 9 days in water phase and less than 1 day in sediment under anaerobic conditions. The DT50 for sediment with low organic carbon content was 20 days in water phase and 18 days in sediment under aerobic conditions and 29 days in water phase and 24 days in sediment under anaerobic conditions (van der Togt and van Ginkel, 2005)

Seawater

Biological transformation of chlorate through assimilatory nitrate reductases is probably the only significant sink for chlorate in the marine environment.The fate of sodium chlorate in seawater is therefore primarily linked to nitrate assimilation rates. In thethe nitrate-nitrogen assimilation rate were <0.2 nM day-1 in the oligotropic period and >2.3 nM day-1 during blooms (Lipchultz, 2001).  Compared to nitrate-nitrogen uptake rates, chlorate reduction rates are assumed to be 10 times lower (expert opinion). The total amount of chlorate in the environment compared to nitrate assimilated is negligible. Chlorate can therefore be completely transformed. Lander et al (1994) measured chlorate concentrations of <24 nM in the Baltic sea at 3-4 km from the effluent discharge of a pulp and paper plant using chlorate.

The environmental chlorate concentration in the oceans is expected to be at least 10 times lower than found by Lander et al. (1994). Using this environmental chlorate concentration for seawater of 2.4 nM, half lives ranging from 5.2 days to 60 days can be calculated (zero order kinetics).

Waste water treatment systems

More than 99% chlorate removal was achieved in a continuously fed up-flow fixed bed reactor operated at a hydraulic retention time of only 3.6 hours. Chlorate was reduced with molasse (Detaille et al 1992). Anaerobic fixed-film processes show that bacteria can remove chlorates from kraft bleach effluent with less than one hour retention time. (Malmqvist and Welander, 1992). Complete biological chlorate removal was also achieved in a pilot-scale bioreactor with suspended carrier material at hydraulic retention times as short as 24 minutes (Malmqvist and Welander, 1994). Pilot studies were carried out with anaerobic (to remove chlorate) and aerobic (to oxidize organic matter) reactors in series. The chlorate concentrations in softwood and hardwood effluents averaged 80 (30-180) and 100 (30-160) mg/L, respectively. The COD concentrations ranged from 1.3 to 2.5 and 0.7 to 1.9 g/L for softwood and hardwood effluents, respectively. In this pilot study chlorate removal percentages of >90 were obtained (Malmqvist and Welander, 1993).   

Chlorate levels of 60 to 70 mg/L were reduced to less than 2 mg/L within a two-hours anaerobic pre-treatment period and eight hours of aerobic treatment in a laboratory scale activated sludge system. A denitrifying culture can not remove chlorate under these conditions (Malyk, 1992).

Laboratory anaerobic and aerobic reactors treating kraft bleach plant effluents operated continuously, removed chlorate easily. Removal of chlorate in softwood and hardwood effluents was 99% and 96%, respectively, with little difference in efficiency between the single-stage and two-stage anaerobic systems (Dorica and Elliot, 1994)