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Environmental fate & pathways

Biodegradation in soil

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Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In order to characterize the catabolism of DCD, different bacteria have been isolated from DCD-treated composts and grown in pure cultures. Two lines have been selected which are able to break down DCD rapidly. DCD metabolism was further followed by thin-layer chromotography (TLC) on silica gel.
GLP compliance:
not specified
Test type:
other: laboratory experiment
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
other: not applicable
% Degr.:
100
Parameter:
test mat. analysis
Sampling time:
3 d
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes

The degradation of dicyandiamide (DCD) by the Mycobacterium smegmatis was very rapid: 200 µg DCD-N/mL were metabolized within 3 days. There was no change of DCD concentrations in the sterile controls. Considerable growth was observed only by DCD-supplied bacteria. Pseudomonas sp. behaved similarly: concomitantly with a rapid decrease in DCD concentration there was rapid bacterial growth.

By TLC it could be shown that there is no decomposition of DCD in the sterile control.

When incubated with Mycobacterium smegmatis, three different degradation products could be seen after 3 days of culture which never appeared in the controls. Pseudomonas sp. also metabolized DCD in 3 days, yielding two metabolites.

Apparently there are at least two different ways of DCD degradation by bacteria. The main metabolites formed by Mycobacterium smegmatis are supposed to be cyanourea which appears as the first metabolite, urea (confirmed by enzymatic testing; Boehringer "Harnstoff-Test"), and a third unidentified product. When incubating DCD with Pseudomonas sp., on the chromatogram a substance together with guanidine and a further, unknown product could be found.

The DCD-degrading principle is heat-labile, as could be shown by boiling for 30s. So far, no buffer extractable DCD-degrading system could be found in Pseudomonas sp.

Conclusions:
The following conclusions can be draw from the experiments:
1) Dicyandiamide (DCD) can be decomposed by soil bacteria.
2) There are at least two different ways of DCD catabolism. Both seen to be different from the inorganic catalytic DCD breakdown with metallic oxides.
In Mycobacterium smegmatis, the possible metabolites are cyanourea, urea an unidentified substance, whereas in Pseudomonas sp. guanidine and another unknown product appear during the degradation.
3) From Mycobacterium smegmatis, a heat-labile substance could be extracted with buffer which is able to decompose DCD readily, suggesting enzymatic control.
Executive summary:

In this experiment it could be shown that apart form abiotic degradation there also exists a biological degradation of dicyandiamide. This has been confirmed with bacteria isolated from lumber compost. To further characterize the catabolism of dicyandiamide, different bacteria have been isolated and grown in pure cultures. Two lines have been selected which are able to break down dicyandiamide rapidly. The first one (line No. 16-1) is Mycobacterium smegmatis, the second one (line No. 11-1) is Pseudomonas sp. The degradation of dicyandiamide by the isolate Mycobacterium smegmatis was very rapid: 200 µg dicyandiamide-N/mL (which corresponds to 300 µg dicyandiamide/mL) were metabolized within 3 days. There was no change of dicyandiamide concentrations in the sterile controls. Considerable growth was observed only by the dicyandiamide-supplied bacteria. Pseudomonas sp. behaved similarly: concomitantly with a rapid decrease in dicyandiamide concentration there was rapid bacterial growth.

Dicyandiamide metabolism was further followed by thin-layer chromatography (TLC) on silica gel. It could be shown that there is no decomposition of dicyandiamide in the sterile control. When incubated with Mycobacterium smegmatis, three different degradation products could be seen after 3 days of culture, which never appeared in the controls. Pseudomonas sp. also metabolized dicyandiamide in 3 days, yielding two metabolites.

Apparently there are at least two different ways of dicyandiamide degradation by bacteria. The main metabolites formed by Mycobacterium smegmatis are supposed to be cyanourea which appears as the first metabolite (unpublished observations), urea (confirmed by enzymatic testing), and a third still unidentified product. When incubating dicyandiamide with Pseudomonas sp., guanidine and a further, unknown product were found. It is notable that Mycobacterium smegmatis and Pseudomonas seem to be unable to metabolize urea (unpublished observations). In contrast to the dicyandiamide degradation with metallic oxides, guanylurea in biological dicyandiamide cleavage was never observed. Cell-free phosphate buffer extracts from Mycobacterium smegmatis were able to degrade dicyandiamide quantitatively into cyanourea at a very high rate. Urea was never detected as a degradation product. The dicyandiamide-degrading principle is heat-labile, as could be shown by boiling for 30 s.

Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1979
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Under different moisture conditions the breakdown of dicyandiamide (DCD) was investigated in quartz sand with and without metal oxides, sandy silty loam, and sand. Degradation was followed by test material analyses (photometric determination).
GLP compliance:
not specified
Test type:
laboratory
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
not specified
Soil no.:
#1
Soil type:
other: quartz sand without metal oxides
Soil no.:
#2
Soil type:
other: quartz sand with metal oxides
Soil no.:
#3
Soil type:
other: sandy silty loam
pH:
6.5
Soil no.:
#4
Soil type:
sand
pH:
6.3
Details on soil characteristics:
SOIL COLLECTION AND STORAGE
- Geographic location: Southern Germany
- Pesticide use history at the collection site: not reported
- Collection procedures: not reported
- Sampling depth (cm): not reported
- Storage conditions: not reported
- Storage length: not reported
- Soil preparation (e.g., 2 mm sieved; air dried etc.): not reported

Soil No.:
#1
Duration:
100 d
Soil No.:
#2
Duration:
40 d
Soil No.:
#3
Duration:
100 d
Soil No.:
#4
Duration:
100 d
Soil No.:
#1
Initial conc.:
ca. 300 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#2
Initial conc.:
ca. 300 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#3
Initial conc.:
ca. 300 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#4
Initial conc.:
ca. 300 mg/kg soil d.w.
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Soil No.:
#1
Temp.:
18 °C
Humidity:
5 %, 50 %, and 150 % of water holding capacity
Microbial biomass:
not determined
Soil No.:
#2
Temp.:
18 °C
Humidity:
5 %, 50 %, and 150 % of water holding capacity
Microbial biomass:
not determined
Soil No.:
#3
Temp.:
18 °C
Humidity:
11 %, 25%, 75 %, and 150 % of water holding capacity
Microbial biomass:
not determined
Soil No.:
#4
Temp.:
18 °C
Humidity:
11 %, 25 %, 75 %, and 150% of water holding capacity
Microbial biomass:
not determined
Details on experimental conditions:
No details reported
Soil No.:
#2
% Recovery:
100
Remarks on result:
other: initial amount: 20 mg DCD-N; sum of remaining DCD-N, guanylurea-N, urea-N, and ammonium-N are approx. 20 mg N (for details see table 1)
Soil No.:
#1
% Degr.:
0
Parameter:
test mat. analysis
Sampling time:
100 d
Soil No.:
#2
% Degr.:
50
Parameter:
test mat. analysis
Sampling time:
5 d
Soil No.:
#2
% Degr.:
90
Parameter:
test mat. analysis
Sampling time:
40 d
Soil No.:
#4
% Degr.:
>= 20 - <= 70
Parameter:
test mat. analysis
Sampling time:
100 d
Soil No.:
#5
% Degr.:
>= 20 - <= 70
Parameter:
test mat. analysis
Sampling time:
100 d
Remarks on result:
other: half-lives were not estimated
Transformation products:
yes
No.:
#1
No.:
#2
Details on transformation products:
- Description of biotransformation pathway: no
- Figure attached: Yes
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes


TRANSFORMATION PRODUCTS
- Guanylurea
- Ammonium

RESULTS:
1) In quartz sand without metal oxides DCD did not change over 100 days. The chemical breakdown of dicyandiamide is catalyzed by metal oxides through adsorption of water and formation of guanylurea. In soils guanylurea was further degraded to ammonium.
2) In the presence of amorphous Fe(III)-hydroxide DCD was transformed to guanylurea after 5 days to 50 % and after 40 days to 90 %. The dicyandiamide-transformation-rate increased with low humidity, and, in the presence of metal oxides, is dependent on the specific surface area of different metal oxides.
3) In two soils (pH 6.5, 6.3; sandy silty loam and sand) breakdown of DCD to guanylurea followed the same pattern, but continued to ammonium. About 20 - 70 % of the added amount was transformed within 100 days.
4) With increasing soil moisture the trasnformation rate of DCD to guanylurea was slower, but the further breakdown to ammonium increased.
5) As long as DCD was present the formation of nitrate was blocked.


FOR DETAILS REFER TO FIGURES 1, 2, and 3 of the attached document
Results with reference substance:
Not applicable -> no reference substance

Table 1: Degradation of dicyandiamide (DCD) in the presence of metal oxides after 10 days (100 g quartz sand + 0.5 g MeO + 20 mg DCD-N, 5 % WK, 18 °C)

Metal oxid

DCD

Guanylurea

Urea

Ammonium

mg N

Fe(III)-hydroxid

6.4

14.0

0.0

0.1

MnO(OH)2

9.9

10.0

0.0

0.0

Cu(OH)2

13.3

6.0

0.0

0.0

Zn(OH)2

18.0

2.0

0.0

0.0

Rust

19.9

0.0

0.0

0.0

Ochre

20.0

0.0

0.0

0.1

Mn(OH)2

20.1

0.0

0.0

0.1

 

Conclusions:
The following conclusions can be drawn from the above mentioned experiments:
1) Chemical breakdown of dicyandiamide is catalyzed by metal oxides.
2) Degradation of dicyandiamide in soils depends on soil moisture.
3) Main degradation products are guanylurea and ammonium.
Executive summary:

Amberger and Vilsmeier (1979) investigated the breakdown of dicyandiamide (20 mg dicyandiamide-N/100 g soil) under different moisture conditions in quartz sand with metal oxides and in soils. They were able to show that

1) In quartz sand without metal oxides DCD did not change over 100 days. The chemical breakdown of dicyandiamide is catalyzed by metal oxides through adsorption of water and formation of guanylurea. In soils guanylurea was further degraded to ammonium.

2) In the presence of amorphous Fe(III)-hydroxide DCD was transformed to guanylurea after 5 days to 50 % and after 40 days to 90 %. The dicyandiamide-transformation-rate increased with low humidity, and, in the presence of metal oxides, is dependent on the specific surface area of different metal oxides.

3) In two soils (pH 6.5, 6.3; sandy silty loam and sand) breakdown of DCD to guanylurea followed the same pattern, but continued to ammonium. About 20 - 70 % of the added amount was transformed within 100 days.

4) With increasing soil moisture the transformation rate of DCD to guanylurea was slower, but the further breakdown to ammonium increased.

5) As long as DCD was present the formation of nitrate was blocked.

Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1980
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A solution with 20 mg DCD-N was added to a sandy silty loam (pH 6.2, N 0.13 %, C 1.09 %) and degradation of DCD was measured after 5, 10, 20, 40, 60, and 100 days of incubation at 10- 90 °C.
GLP compliance:
not specified
Test type:
other: laboratory and field trails
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
not specified
Soil no.:
#1
Soil type:
other: Sandy silty loam
pH:
6.2
Soil No.:
#1
Duration:
>= 5 - <= 100 d
Soil No.:
#1
Initial conc.:
ca. 300 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#1
Initial conc.:
10 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#1
Initial conc.:
20 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#1
Temp.:
10-90 °C
Humidity:
40 - 50 % of water holding capacity
Microbial biomass:
not determined
Soil No.:
#1
% Degr.:
>= 14 - <= 100
Parameter:
test mat. analysis
Sampling time:
20 d
Transformation products:
yes
No.:
#1
No.:
#2
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes
Conclusions:
The following conclusions can be drawn from the above mentioned experiments:
1) Degradation of dicyandiamide in soils strongly depends on soil temperature.
2) Main degradation products are guanylurea, ammonium.
Executive summary:

Vilsmeier (1980) examined the degradation of dicyandiamide in soil in relation to temperature (soil #1).

They applied 20 mg dicyandiamide-N/100 g soil. At temperatures of 10-90 °C dicyandiamide was metabolized to 14-100 % after 14 days. Small amounts of dicyandiamide (0.67 and 1.34 mg dicyandiamide-N/100g soil) were broken down completely within 20-80 days at 8-20 °C. Vilsmeier was able to clearly demonstrate that dicyandiamide degradation rises with increasing temperature.

   

The following conclusions can be drawn from the above mentioned experiments:

1) Degradation of dicyandiamide in soils depends on soil moisture and temperature.

5) Main degradation products are guanylurea, ammonium.

Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Amberger & Vilsmeier (1988) examined leaching of dicyandiamide after mineral fertilizing and slurry manuring and decomposition of dicyandiamide in flooded soils (simulated ground water conditions; silty loam, pH 6.5). The experiments were conducted at 5-12 °C for 60 weeks with an initial DCD content of 20 mg DCD/L water (300 g wet soil + 600 ml water) at aerobic and anaerobic conditions.
After mineral feeding, only 0.6-0.9 % of dicyandiamide applied in 5 years was leached.
Biodegradation of dicyandiamide was followed by test material analyses (HPLC).
GLP compliance:
not specified
Test type:
other: laboratory and field trails
Radiolabelling:
no
Oxygen conditions:
aerobic/anaerobic
Soil classification:
not specified
Soil no.:
#1
Soil type:
other: Sediment
% Clay:
15
pH:
7
Soil no.:
#2
Soil type:
other: Sediment
% Clay:
10
pH:
7.1
Soil no.:
#3
Soil type:
other: Sediment
% Clay:
5
pH:
7.2
Soil no.:
#4
Soil type:
other: Sediment
% Clay:
6
pH:
7.2
Soil no.:
#5
Soil type:
other: groundwater-influenced soil
% Clay:
8
pH:
7.5
Soil no.:
#6
Soil type:
other: groundwater-influenced soil
% Clay:
5
pH:
7.6
Soil no.:
#7
Soil type:
other: farmland
% Clay:
39
pH:
5.2
Soil no.:
#8
Soil type:
other: farmland
% Clay:
21
pH:
6.1
Soil No.:
#1
Duration:
60 wk
Soil No.:
#2
Duration:
60 wk
Soil No.:
#3
Duration:
60 wk
Soil No.:
#4
Duration:
60 wk
Soil No.:
#5
Duration:
60 wk
Soil No.:
#6
Duration:
60 wk
Soil No.:
#7
Duration:
60 wk
Soil No.:
#8
Duration:
60 wk
Soil No.:
#1
Initial conc.:
ca. 20 other: mg/L water
Based on:
test mat.
Soil No.:
#2
Initial conc.:
20 other: mg/L water
Based on:
test mat.
Soil No.:
#3
Initial conc.:
20 other: mg/L water
Based on:
test mat.
Soil No.:
#4
Initial conc.:
20 other: mg/L water
Based on:
test mat.
Soil No.:
#5
Initial conc.:
20 other: mg/L water
Based on:
test mat.
Soil No.:
#6
Initial conc.:
20 other: mg/L water
Based on:
test mat.
Soil No.:
#7
Initial conc.:
20 other: mg/L water
Based on:
test mat.
Soil No.:
#8
Initial conc.:
20 other: mg/L water
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Soil No.:
#1
Temp.:
5-12 °C
Humidity:
flooded sediment
Microbial biomass:
not determined
Soil No.:
#2
Temp.:
5-12 °C
Humidity:
flooded sediments
Microbial biomass:
not determined
Soil No.:
#3
Temp.:
5-12 °C
Humidity:
flooded sediment
Microbial biomass:
not determined
Soil No.:
#4
Temp.:
5-12 °C
Humidity:
flooded sediment
Microbial biomass:
not determined
Soil No.:
#5
Temp.:
5-12 °C
Humidity:
flooded soil
Microbial biomass:
not determined
Soil No.:
#6
Temp.:
5-12 °C
Humidity:
flooded soil
Microbial biomass:
not determined
Soil No.:
#7
Temp.:
5-12 °C
Humidity:
flooded soil
Microbial biomass:
not determined
Soil No.:
#8
Temp.:
5-12 °C
Humidity:
flooded soil
Microbial biomass:
not determined
Details on experimental conditions:
No details reported
Soil No.:
#1
% Degr.:
100
Parameter:
test mat. analysis
Sampling time:
34 wk
Soil No.:
#2
% Degr.:
100
Parameter:
test mat. analysis
Sampling time:
34 wk
Soil No.:
#3
% Degr.:
96
Parameter:
test mat. analysis
Sampling time:
34 wk
Soil No.:
#4
% Degr.:
100
Parameter:
test mat. analysis
Sampling time:
34 wk
Soil No.:
#5
% Degr.:
96
Parameter:
test mat. analysis
Sampling time:
34 wk
Soil No.:
#6
% Degr.:
94.5
Parameter:
test mat. analysis
Sampling time:
34 wk
Soil No.:
#7
% Degr.:
70.5
Parameter:
test mat. analysis
Sampling time:
34 wk
Soil No.:
#8
% Degr.:
100
Parameter:
test mat. analysis
Sampling time:
34 wk
Transformation products:
not measured
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes

Aerobic conditions:

- total degradation of dicyandiamide within 34 or 44 weeks

Anaerobic conditions:

- 6 -20 % of dicyandiamide were not degraded after 60 weeks of incubation

Table 1: Degradation of dicyandiamide in flooded soils under aerobic and anaerobic conditions (300 g wet soil + 600 ml water)

 

Dicyandiamide (mg/L) after weeks

20

34

44

60

Soil

aerobic

anaerobic

aerobic

anaerobic

aerobic

anaerobic

aerobic

anaerobic

#1

Sediment

14.1

13.4

0

11.3

0

10.6

0

8.2

#2

Sediment

15.5

13.7

0

9.7

0

6.8

0

2.1

#3

Sediment

17.0

15.5

0.8

14.6

0

11.7

0

6.3

#4

Sediment

12.4

11.5

0

8.2

0

0.2

0

0

#5

Groundwater-influenced soil

19.0

16.8

0.8

17.3

0

16.1

0

13.1

#6

Groundwater-influenced soil

16.0

15.0

1.1

14.2

0

13.0

0

9.7

#7

Farmland

18.8

15.8

5.9

14.2

0

13.3

0

7.8

#8

Farmland

18.1

12.5

0

10.9

0

8.7

0

6.3

 

Conclusions:
The following conclusions can be drawn from the above mentioned experiments:
1) Chemical breakdown of dicyandiamide is catalyzed by metal oxides
2) Dicyandiamide can be decomposed by soil bacteria
3) There are at least two different ways of dicyandiamide catabolism. Both seem to be different from the inorganic catalytic dicyandiamide breakdown with metallic oxides. In Mycobacterium smegmatis, the possible metabolites are cyanourea, urea, an unidentified substance, whereas in Pseudomonas sp. guanidine and another unknown product appear during the degradation.
4) Degradation of dicyandiamide in soils depends on soil moisture and temperature
5) Main degradation products are guanylurea, ammonia and nitrate (nitrate-formation only in the absence of dicyandiamide which is a known inhibitor for ammonium oxidation)
Executive summary:

Amberger & Vilsmeier (1988) examined leaching of dicyandiamide after mineral fertilizing and slurry manuring and decomposition of dicyandiamide in flooded soils (simulated ground water conditions; silty loam, pH 6.5). The experiments were conducted at 5-12 °C for 60 weeks with an initial DCD content of 20 mg DCD/L water (300 g wet soil + 600 ml water) at aerobic and anaerobic conditions.

After mineral feeding, only 0.6-0.9 % of dicyandiamide applied in 5 years was leached.

Highest leaching rates od DCD occurred after slurry application in October (with 5.6 % of added amount).

In sediment flooded with water to a height of 10 to 60 cm, dicyandiamide (20 mg/l) was fully degraded within one year in almost all experiments at aerobic conditions while at anaerobic conditions two thirds were decomposed.

Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In order to characterize the catabolism of dicandiamide (DCD), different bacteria have been isolated from DCD-treated composts and grown in pure cultures. Two lines have been selected which are able to break down DCD rapidly. DCD metabolism was further followed by thin-layer chromotography (TLC) on silica gel.
GLP compliance:
not specified
Test type:
other: laboratory experiment
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
other: not applicable
% Degr.:
100
Parameter:
test mat. analysis
Sampling time:
3 d
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
yes

The degradation of dicyandiamide (DCD) by Rhodococcus sp. was very rapid: 200 µg DCD-N/mL were metabolized within 3 days. There was no change of DCD concentrations in the sterile controls. Considerable growth was observed only by DCD-supplied bacteria. Pseudomonas sp. behaved similarly: concomitantly with a rapid decrease in DCD concentration there was rapid bacterial growth.

By TLC it could be shown that there is no decomposition of DCD in the sterile control.

When incubated with Rhodococcus sp., three different degradation products could be seen after 3 days of culture which never appeared in the controls. Pseudomonas sp. also metabolized DCD in 3 days, yielding two metabolites.

Apparently there are at least two different ways of DCD degradation by bacteria. The main metabolites formed by Rhodococcus sp. are supposed to be cyanourea which appears as the first metabolite, urea (confirmed by enzymatic testing; Boehringer "Harnstoff-Test"), and a third unidentified product. When incubating DCD with Pseudomonas sp., on the chromatogram a substance together with guanidine and a further, unknown product could be found. Rhodococcus and Pseudomonas seem to be unable to metabolize urea.

In contrast to the DCD degradation with metallic oxides, guanylurea could never be observed in biological DCD cleavage.

Cell-free phosphate buffer extracts from Rhodococcus sp. were able to degrade DCD quantitatively into cyanourea at a very high rate. Urea was never detected as a degradation product. The DCD-degrading principle is heat-labile, as could be shown by boiling for 30s. So far, no buffer extractable DCD-degrading system could be found in Pseudomonas sp.

Conclusions:
The following conclusions can be draw from the experiments:
1) Dicyandiamide (DCD) can be decomposed by soil bacteria.
2) There are at least two different ways of DCD catabolism. Both seem to be different from the inorganic catalytic DCD breakdown with metallic oxides.
In Rhodococcus, the possible metabolites are cyanourea, urea an unidentified substance, whereas in Pseudomonas sp. guanidine and another unknown product appear during the degradation.
3) From Rhodococcus, a heat-labile substance could be extracted with buffer which is able to decompose DCD readily, suggesting enzymatic control.
Executive summary:

In this experiment it could be shown that apart form abiotic degradation there also exists a biological degradation of dicyandiamide. To further characterize the catabolism of dicyandiamide, different bacteria have been isolated from dicyandiamide-treated composts and were grown in pure cultures. Two lines have been selected which are able to break down dicyandiamide rapidly. The first one (line No. 16-1) is likely to belong to the genus Rhodococcus, the second one (line No. 11-1) is presumably a Pseudomonas sp. The degradation of dicyandiamide by the isolate Rhodococcus was very rapid: 200 µg dicyandiamide-N/mL (which corresponds to 300 µg dicyandiamide/mL) were metabolized within 3 days. There was no change of dicyandiamide concentrations in the sterile controls. Considerable growth was observed only by the dicyandiamide-supplied bacteria. Pseudomonas sp. behaved similarly: concomitantly with a rapid decrease in dicyandiamide concentration there was rapid bacterial growth.

Dicyandiamide metabolism was further followed by thin-layer chromatography (TLC) on silica gel. It could be shown that there is no decomposition of dicyandiamide in the sterile control. When incubated with Rhodococcus sp., three different degradation products could be seen after 3 days of culture, which never appeared in the controls. Pseudomonas sp. also metabolized dicyandiamide in 3 days, yielding two metabolites.

Apparently there are at least two different ways of dicyandiamide degradation by bacteria. The main metabolites formed by Rhodococcus are supposed to be cyanourea which appears as the first metabolite (unpublished observations), urea (confirmed by enzymatic testing), and a third still unidentified product. When incubating dicyandiamide with Pseudomonas sp., guanidine and a further, unknown product were detected. It is notable that Rhodococcus and Pseudomonas seem to be unable to metabolize urea (unpublished observations). In contrast to the dicyandiamide degradation with metallic oxides, guanylurea in biological dicyandiamide cleavage was never observed. Cell-free phosphate buffer extracts from Rhodococcus were able to degrade dicyandiamide quantitatively into cyanourea at a very high rate. The dicyandiamide-degrading principle is heat-labile, as could be shown by boiling for 30 s.

Endpoint:
biodegradation in soil, other
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:
- Principle of test: The influence of oxygen content on the biodegradation of dicyandiamide was assessed
- Short description of test conditions: Soils from two sites with different organic matter concentrations but the same parent material were sampled to the same depth, sieved, and repacked into tubes (‘soil cores’). These were saturated with a DCD solution (30 mg/mL) and placed under controlled aeration conditions by imposing five levels of matric potential (0, –1, –3, –6, and –10 kPa) at a constant temperature (22°C).
- Parameters analysed / observed: The relative O2 diffusivity (O2 diffusion coefficient in soil/O2 diffusion coefficient in air, Dp/Do) was measured, along with periodic destructive sampling of soil cores over 40 days, to assess the DCD concentrations.
GLP compliance:
not specified
Remarks:
Not mentioned in the published literature.
Test type:
laboratory
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
other: Hewitt AE (1998) ‘New Zealand Soil Classification.’ Landcare Research Science Series, No. 1. 2nd edn. (Manaaki Whenua Press: Lincoln, NZ)
Soil no.:
#1
Soil type:
silt loam
% Clay:
19
% Silt:
48
% Sand:
33
% Org. C:
5.34
pH:
5.25
Bulk density (g/cm³):
1.1
Soil no.:
#2
Soil type:
silt loam
% Clay:
19
% Silt:
48
% Sand:
33
% Org. C:
3.44
pH:
5.03
Bulk density (g/cm³):
1.1
Details on soil characteristics:
SOIL COLLECTION AND STORAGE
- Geographic location: Silt loam soil (0–15 cm) from a tillage trial, Lincoln Zealand (43840003.9100S, 172828011.7600E 5 m a.s.l.)
- Pesticide use history at the collection site: Soil was collected from two sites (A and B) within this trial, which, for the past 14 years, had differing cropping histories. Site A was a control plot maintained under a grass sward, whereas site B was a permanent fallow.
- Collection procedures: not reported
- Sampling depth (cm): 0-15 cm
- Storage conditions: not reported
- Storage length: not reported
- Soil preparation (e.g., 2 mm sieved; air dried etc.): Soil from each site was air dried and sieved to <2 mm. Soil cores were then constructed by packing sieved soil into stainless-steel rings (internal diameter 7.3 cm) to a bulk density of 1.1 g/cm³ and depth of 4.1 cm.

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm (%): not reported
- Bulk density (g/cm3):1.1
Soil No.:
#1
Duration:
40 d
Soil No.:
#2
Duration:
40 d
Soil No.:
#1
Initial conc.:
>= 6.6 - <= 11.1 mg/kg soil d.w.
Based on:
test mat.
Soil No.:
#2
Initial conc.:
>= 6.6 - <= 11.1 mg/kg soil d.w.
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on experimental conditions:
1. PRELIMINARY EXPERIMENTS:
Not reported
2. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): no
- Soil condition: air dried
- Soil (g/replicate): stainless-steel rings (internal diameter 7.3 cm) filled with sieved soil to a bulk density of 1.1 g/cm³ and depth of 4.1 cm  ca 188.8 g (d.w.) per replicate
- Control conditions, if used (present differences from other treatments, i.e., sterile/non-sterile, experimental conditions): control cores were saturated with deionised water
- No. of replication controls, if used: 4 replicates in total 64 control soil cores; Thirty-two control soil cores (2 sites * 4 levels of matric potential* 4 replicates) were destructively sampled on both day 1 and day 40.
- No. of replication treatments: 4 replicates per treatment plus additional replicates for 8 time points of analytical measurements:
232 in total -8 for intial measurement =224 (Eight of the DCD-treated soil cores (four replicates of each soil, A and B) were destructively sampled on day 0 to measure soil DCD concentrations immediately after saturation.)
The remaining DCD-treated soil cores (224) were subsampled on days 1, 4, 8, 12, 20, 30 and 40 by taking 32 soil cores (2 sites * 4 levels of matric potential* 4 replicates).
- Test apparatus (Type/material/volume): Soil cores were arranged in a randomised block design on tension tables. Before placing soil cores on the tension tables at the designated y levels, 232 soil cores were pre-saturated with a DCD solution (30 mg/mL) to obtain soil DCD concentrations, following 24-h drainage, ranging from 6.6 to 11.1 mg/kg soil.
- Details of traps for CO2 and organic volatile, if any: no
- If no traps were used, is the system closed: closed
- Identity and concentration of co-solvent: water

Test material application
- Volume of test solution used/treatment: not reported
- Application method (e.g. applied on surface, homogeneous mixing etc.): soil cores were pre-saturated with a DCD solution (30 mg/mL) and drained after 24 hours
- Is the co-solvent evaporated: test material dissolved in water, not evaporated but drained

Any indication of the test material adsorbing to the walls of the test apparatus: no

Experimental conditions (in addition to defined fields)
- Moisture maintenance method:
- Continuous darkness: not reported

Other details, if any: Measurement of pore size: To determine the pore-size distribution, a water-retention curve (WRC) was fitted to a plot of volumetric water content (Ɵ, cm³/cm³) v. matric potential Ψ (kPa). Including logarithmic transformation followed by linear regression, the value of parameter b was determined according to the following equation:
(Equation 1): Ψ=Ψe (Ɵ/Ɵs)^-b
where Ψe is the air-entry value and Ɵs is the saturated water content. Equation 1 was combined with capillary rise equation, Ψ= –2γ/r, where γ is a ratio of water’s surface tension and density, and r is an ‘effective’ or ‘narrowest channel’ pore radius, to estimate four values of r associated with decreasing Ψ from saturation (Ψ= 0) through each of the four levels applied as treatments.

3. OXYGEN CONDITIONS (delete elements as appropriate)
- Methods used to create the aerobic conditions: The soil samples were placed under controlled aeration conditions by imposing five levels of matric potential (0, –1, –3, –6, and –10 kPa) at a constant temperature (22°C).
- Evidence that aerobic conditions were maintained during the experiment (e.g. redox potential): Relative O2 diffusivity (O2 diffusion coefficient in soil/O2 diffusion coefficient in air, Dp/Do) was measured. A calibrated O2 sensor (KE-25; Figaro Engineering Inc., Osaka, Japan) was placed in a chamber that was then purged until anaerobic by using an Ar (90%) and N2 (10%) gas mixture, with the base of the soil core isolated from the chamber. Once anaerobic, the soil core was exposed to the O2-free chamber atmosphere. Oxygen diffusing through the soil core into the chamber was recorded as a function of change in concentration over time. Consumption of O2 was considered negligible. Regression analysis of the log-plot of relative O2 concentration v. time enabled Dp (O2 diffusion coefficient in soil) to be calculated according to Rolston and Moldrup (Rolston DE, Moldrup P (2002) Gas diffusivity. In ‘Methods of soil analysis, Part 4, Physical methods’. (Eds GC Topp, JH Dane) pp. 1113–1139. Soil Science Society of America: Madison, WI, USA). All diffusivity calculations were performed at 25°C and the value of Do (O2 diffusion coefficient in air) at 25 °C was calculated to be 0.074 m2/h. Relative gas diffusivity was expressed as Dp/Do. According to the method of Rolston and Moldrup (2002), described by Balaine et al. (2013).

4. SUPPLEMENTARY EXPERIMENTS: /

5. SAMPLING DETAILS
- Sampling intervals: days 0, 1, 4, 8, 12, 20, 30 and 40 by taking 32 soil cores (2 sites * 4 levels of matric potential * 4 replicates).
- Sampling method for soil samples: destructively sampled, therefore additional measurement replicates were prepared: in total 296 (including controls)
- Method of collection of CO2 and volatile organic compounds: not conducted
- Sampling intervals/times for: n.a.
> Sterility check, if sterile controls are used: n.a.
> Moisture content: not reported
> Redox potential/other: not reported
> Sample storage before analysis: not reported
- Other observations, if any: no
Soil No.:
#1
DT50:
15.4 d
Type:
(pseudo-)first order (= half-life)
Temp.:
22 °C
Remarks on result:
other: matric potential: -10 kPa
Soil No.:
#1
DT50:
16.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
22 °C
Remarks on result:
other: matric potential: -6.0 kPa
Soil No.:
#1
DT50:
21 d
Type:
(pseudo-)first order (= half-life)
Temp.:
22 °C
Remarks on result:
other: matric potential: -3.0 kPa
Soil No.:
#1
DT50:
27.6 d
Type:
(pseudo-)first order (= half-life)
Temp.:
22 °C
Remarks on result:
other: matric potential: -1.0 kPa
Soil No.:
#2
DT50:
22.4 d
Type:
(pseudo-)first order (= half-life)
Temp.:
22 °C
Remarks on result:
other: matric potential:-10 kPa
Soil No.:
#2
DT50:
23.1 d
Type:
(pseudo-)first order (= half-life)
Temp.:
22 °C
Remarks on result:
other: matric potential:-6.0 kPa
Soil No.:
#2
DT50:
24.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
22 °C
Remarks on result:
other: matric potential:-3.0 kPa
Soil No.:
#2
DT50:
31.5 d
Type:
(pseudo-)first order (= half-life)
Temp.:
22 °C
Remarks on result:
other: matric potential: -1.0 kPa
Transformation products:
not measured
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
not measured
Details on results:
TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): No

MAJOR TRANSFORMATION PRODUCTS
- not measured

MINOR TRANSFORMATION PRODUCTS
- not measured

EXTRACTABLE RESIDUES
- % of applied amount at day 0: Not measured
- % of applied amount at end of study period: Not measured
- No DCD was detected in the controls. Initial DCD concentrations in soil cores receiving DCD, measured on days 0 and 1, along with the respective volumetric water content Ɵ values are provided in Table 1.
DCD recovery from both sites was estimated in order to check for adsorption due to variation in soil organic matter content. Estimated DCD recovery was 91.2% for soil A with 5.34% organic matter and 92.8% for soil B with 3.44% organic matter.
Concentrations of DCD in soil from both sites decreased exponentially with time from day 4 to day 40 for all levels of matric potential (Fig. 1a, b). Higher DCD concentrations (P <0.05) were observed under wetter soil conditions (–1.0 kPa) throughout the measurement period.

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: Not measured
- % of applied amount at end of study period: Not measured

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: Not measured

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: Not measured

Values of Ɵ from day 4 to day 40 remained constant for soil from both sites. Mean (and standard deviation) values of Ɵ for site A at –10, –6.0, –3.0 and –1.0 kPa were 0.24 (0.01), 0.30 (0.01), 0.37 (0.02) and 0.47 (0.02) cm3/cm3, respectively; for site B, corresponding values were 0.25 (0.01), 0.30 (0.01), 0.36 (0.01) and 0.47 (0.02) cm³/cm³, with no difference due to site.

Because Ɵ did not differ with soil site, the WRC was fitted by using data from both sites (Table 2). Fitting Equation 1 to the WRC data yielded values for Ψe and b of 0.055 ± 0.009 and –3.514 ± 0.447, respectively. Pore-size distribution showed that 33 % of the soil volume had pores with an ‘effective’ diameter >30 mm, 6% 30–50 mm, 7% 50–100 mm, 9% 100–300 mm, and 11% >300 mm.

Values of the estimated parameters (C0and k) derived from the regression analysis and measured Dp/Do values at different Ψ levels are shown in Table 3. For each site, the soil matric potential (Ψ) affected the degradation (k) constant, with lower values at –1.0 kPa (P <0.05). When values of k were compared between sites A and B for each level of Ψ, values were higher (P <0.05) for site A at –10 and –6.0 kPa (Fig. 2). Thus, the calculated half-life of DCD at site A increased as soil became wetter, with values (mean ± standard error) of 15.4 ± 2.4, 16.9 ± 2.8, 21.0 ± 3.4, and 27.6 ± 3.5 days at –10, –6.0, –3.0 and –1.0 kPa, respectively. For soil from site B, the DCD half-life also increased as soil became wetter with corresponding values of 22.4 (±5.8), 23.1 (±4.4), 24.7 (±4.8), 31.5 (±5.5) days. Measured values of Dp/Do increased as matric potential became more negative; however, Dp/Do was not affected by soil site (Table 3).

 

Table 2: Soil DCD concentrations and volumetric water contents (Ɵ) measured on day 0 and after 1 day at varying matric potentials. Values in parentheses are standard deviations (n = 4).

 

Day 0

Day 1

 

 

-10 kPa

-6.0 kPa

-3.0 kPa

-1.0 kPa

 

mg DCD /kg soil

Site A

14.84

(0.86)

7.08

(0.28)

8.41

(0.13)

9.40

(0.33)

11.10

(0.38)

Site B

15.12

(1.21)

6.63

(0.21)

8.53

(0.34)

9.99

(0.38)

10.78

(0.34)

 

Ɵ (m³/m³)

Site A

0.56

(0.10)

0.37

(0.01)

0.42

(0.01)

0.44

(0.01)

0.51

(0.02)

Site B

0.52

(0.02)

0.36

(0.01)

0.41

(0.01)

0.44

(0.01)

0.50

(0.01)

 

Table 3: Volumetric soil water content (Ɵ) at five levels of matric potential (Ψ) and the corresponding ‘effective’ pore diameter drained, as explained in the text. The values were not significantly different for Soil A and B.

Ψ (kPa)

Ɵ (m³/m³)

Pore diameter drained (µm)

0

0.57

–1.0

0.46

300

–3.0

0.37

100

–6.0

0.30

50

–10

0.24

30

 

Table 4: Regression parameters (C0and k) and measured Dp/Do values for soil from sites A and B. Values in parentheses are standard deviations for Dp/Do, and standard errors of the estimate derived from the regressions performed in Fig. 1 for C0and k. Within a column, means followed by the same letter are not significantly different at P = 0.05.

 

Soil A

Soil B

Matric potential (kPa)

C0(mg/kg)

k (day-1)

Dp/Do

C0(mg/kg)

k (day-1)

Dp/Do

–10

7.43a (0.22)

0.045a (0.002)

0.07a (0.008)

6.96a (0.27)

0.031a (0.003)

0.08a (0.005)

–6.0

8.69b (0.26)

0.041a (0.002)

0.03b (0.004)

7.65b (0.24)

0.029a (0.002)

0.03b (0.003)

–3.0

8.90b (0.24)

0.033b (0.002)

0.01c (0.003)

9.03c (0.24)

0.029a (0.002)

0.01c (0.002)

–1.0

11.45c (0.22)

0.025c (0.001)

0d

10.52d (0.23)

0.022b (0.001)

0d

 

Table 5: Calculated half-life of DCD. Half-life increased with decreased matric potential.

 

t1/2(days)

Matric potential (kPa)

Soil A

Soil B

–10

15.4 ± 2.4

22.4 ± 8.8

–6.0

16.9 ± 2.8

23.1 ± 4.4

–3.0

21.0 ± 3.4

24.7 ± 4.8

–1.0

27.6 ± 3.5

31.5 ± 5.5

 

These values show that the matric potential influences the degradation of dicyandiamide in the soil. The present study also showed that measured DCD degradation rates became lower as Dp/Do - a measure of soil aeration - decreased. When DCD is applied to well-aerated soils, its lifetime will be shorter than in wet soils, all other factors being constant. Differences between soils taken from sites A and B included higher pH, soil carbon and organic matter at site A (Table 1), which potentially resulted in differences in soil microbial community structure and function, nutrient availability and microbial biomass.

Conclusions:
The calculated half-life of DCD in soil A varied from 15.4 to 27.6 days at 22°C in dependence of the matric potential.
For soil B the calculated half-life ranged from 22.4 to 31.5 days at 22 °C in dependence of the matric potential.
Executive summary:

The biotransformation of dicyandiamide (DCD) was studied in silt loam soil from a control plotmaintained under a grass sward (Soil A) (pH 5.25, organic matter 5.34 %) and silt loam soil from a permanent fallow (Soil B) (pH 5.03, organic matter 3.44 %) from Lincoln, New Zealand for 40 d under aerobic conditions in dark at 22 ºC, and matric potential of –10, –6.0, –3.0, –1.0. kPa. Soil cores were pre-saturated with a DCD solution (30 mg mL–1) to obtain soil DCD concentrations, following 24-h drainage, ranging from 6.6 to 11.1 mg/kgsoil. The test system consisted of stainless-steel rings packed with soil not attached with traps for the collection of CO2 and volatile organics.  Samples were analysed at 0, 1, 4, 8, 12, 20, 30 and 40 of incubation.  The soil samples were extracted with water, and the DCD residues were analysed by HPLC-UV.  Identification of the transformation products was not conducted.

Calculated half-life of DCD. Half-life increased with decreased matric potential.

 

t1/2(days) at 22°C

Matric potential (kPa)

Soil A

Soil B

–10

15.4 ± 2.4

22.4 ± 8.8

–6.0

16.9 ± 2.8

23.1 ± 4.4

–3.0

21.0 ± 3.4

24.7 ± 4.8

–1.0

27.6 ± 3.5

31.5 ± 5.5

 

The presented data show that the matric potential influences the degradation of dicyandiamide in the soil. The present study also showed that measured DCD degradation rates became lower as Dp/Do - a measure of soil aeration - decreased. When DCD is applied to well-aerated soils, its lifetime will be shorter than in wet soils, all other factors being constant. Differences between soils taken from sites A and B included higher pH, soil carbon and organic matter at site A (Table 1), which potentially resulted in differences in soil microbial community structure and function, nutrient availability and microbial biomass.

Endpoint:
biodegradation in soil, other
Type of information:
other: Based on published data from controlled-environment studies of soils sampled in four countries, the present study modelled the relation between T and the time for DCD concentration in soils to decline to half its application value.
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline followed
Principles of method if other than guideline:
Compilation and analysis of the published data relating to the temperature dependence of DCD degradation in soils with regards to its effectiveness in reducing N2O emissions.
GLP compliance:
no
Test type:
other: Calculation
Radiolabelling:
no
Oxygen conditions:
not specified
Soil classification:
not specified
Year:
2008
Soil no.:
#1
Soil type:
other: no data
Soil no.:
#2
Soil type:
silt loam
% Org. C:
0.73
pH:
6.1
Soil no.:
#3
Soil type:
sandy loam
% Org. C:
0.73
pH:
7.5
Bulk density (g/cm³):
1.3
Soil no.:
#4
Soil type:
silt loam
% Org. C:
3.65
pH:
5.9
Details on soil characteristics:
Soil no# 1: no data
Soil no# 2: Giessen, Germany: Silt loam, 29 g organic carbon/kg, 2.5 g total nitrogen/kg, pH: 6.1; source: Rajbanshi et al. 1992.
Soil no# 3: Navarra, Spain: Sandy loam, 7.3 g organic carbon/kg, 1.3 g total nitrogen/kg, pH 7.5; bulk density: 1.3 mg/m³;application of dicyandiamide over a soil depth of 0.1 m; source: Irigoyen et al. 2003a.
Soil no# 4: Lincoln, New Zealand: Silt loam, 36.5 g organic carbon/kg, 3.5 g total nitrogen/kg, pH: 5.9; source: Di and Cameron 2004.
Soil No.:
#2
Initial conc.:
13 kg/ha d.w.
Based on:
test mat.
Soil No.:
#2
Initial conc.:
32 kg/ha d.w.
Based on:
test mat.
Soil No.:
#3
Initial conc.:
5.2 kg/ha d.w.
Based on:
test mat.
Soil No.:
#4
Initial conc.:
7.5 kg/ha d.w.
Based on:
test mat.
Parameter followed for biodegradation estimation:
not specified
Soil No.:
#1
Temp.:
0,6, and 12 °C
Humidity:
not reported
Microbial biomass:
not reported
Soil No.:
#2
Temp.:
10, 20, and 30 °C
Humidity:
maintained near the soil’s field capacity
Microbial biomass:
not reported
Soil No.:
#3
Temp.:
8, and 20
Humidity:
maintained near the soil’s field capacity
Microbial biomass:
not reported
Details on experimental conditions:
The water content of incubated samples was maintained near the soil’s field capacity. Consequently, the sandy soil of Irigoyen et al. (2003) was kept significantly drier than the silt loam soils (Di and Cameron, 2004; Rajbanshi et al., 1992).
Soil No.:
#1
DT50:
98 d
Type:
(pseudo-)first order (= half-life)
Temp.:
6 °C
Remarks on result:
other: k = 0.0071 per day
Soil No.:
#1
DT50:
147 d
Type:
(pseudo-)first order (= half-life)
Temp.:
0 °C
Soil No.:
#1
DT50:
42 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Soil No.:
#2
DT50:
52 d
Type:
zero order
Temp.:
10 °C
Remarks on result:
other: 13 kg DCD/ha applied
Soil No.:
#2
DT50:
16 d
Type:
zero order
Temp.:
20 °C
Remarks on result:
other: 13 kg DCD/ha applied
Soil No.:
#2
DT50:
13 d
Type:
zero order
Temp.:
30 °C
Remarks on result:
other: 13 kg DCD/ha applied
Soil No.:
#2
DT50:
70 d
Type:
zero order
Temp.:
10 °C
Remarks on result:
other: 32 kg DCD/ha applied
Soil No.:
#2
DT50:
18 d
Type:
zero order
Temp.:
20 °C
Remarks on result:
other: 32 kg DCD/ha applied
Soil No.:
#2
DT50:
12 d
Type:
zero order
Temp.:
30 °C
Remarks on result:
other: 32 kg DCD/ha applied
Soil No.:
#3
DT50:
> 105 d
Type:
zero order
Temp.:
10 °C
Remarks on result:
other: 5.2 kg DCD/ha applied
Soil No.:
#3
DT50:
18 d
Type:
zero order
Temp.:
20 °C
Remarks on result:
other: 5.2 kg DCD/ha applied
Soil No.:
#3
DT50:
7 d
Type:
zero order
Temp.:
30 °C
Remarks on result:
other: 5.2 kg DCD/ha applied
Soil No.:
#4
DT50:
111 d
Type:
(pseudo-)first order (= half-life)
Temp.:
8 °C
Remarks on result:
other: 7.5 kg DCD/ha applied
Soil No.:
#4
DT50:
26 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: 7.5 kg DCD/ha applied
Soil No.:
#4
DT50:
116 d
Type:
(pseudo-)first order (= half-life)
Temp.:
8 °C
Remarks on result:
other: 15 kg DCD/ha applied
Soil No.:
#4
DT50:
18 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Remarks on result:
other: 15 kg DCD/ha applied
Transformation products:
not specified
Evaporation of parent compound:
not specified
Volatile metabolites:
not specified
Residues:
not specified
Details on results:
A first-order model was fitted to a plot of t½ and soil temperature (T). The synthesis analysis included 16 measurements (see Table 1) from four studies (Amberger, 1989; Di and Cameron, 2004; Irigoyen et al., 2003; Rajbanshi et al., 1992; i.e. soil no# 1 to 4).

Table 1: Overview of the data presented in the review.

Authors

Experimental data for calculation model

Modelled half-life in days

Temperature in °C

Medium/soil type

Amount of DCD in kg DCD/ha

Location

Texture

Organic carbon in g/kg

Total nitrogen in g/kg

pH

Hauser and Haselwandter (1990)

25

Nutrient solution and soil bacteria strain EK1

Unknown

9 *

Amberger 1989

6

No data

 

Unspecified

 

98 *

109 **

0

147*

12

42 *

Bronson et al. 1989

8

Alabama USA

Silt loam

 

8.0

No data

 

6.2

130

26 **

15

130

13 **

22

130

8 **

Rajbanshi et al. 1992

10

Giessen. Germany

Silt loam

 

29.0

2.5

6.1

13

52 **

20

13

16 **

30

13

13 **

10

32

70 **

20

32

18 **

30

32

12 **

20

65

22 **

30

65

15 **

10

Giessen. Germany

Silt loam

 

29.0

2.5

6.1

Pretreatment with DCD + 13 kg DCD ha-1

7#

10

Pretreatment with DCD + 13 kg DCD ha-1

13#

Irigoyen et al. 2003a)

10

Navarra. Spain

Sandy loam

7.3

1.3

7.5

5.2

> 105 **

20

5.2

18 **

30

5.2

7 **

Di and Cameron 2004

 

 

8

Lincoln. New Zealand

Silt loam

36.5

3.5

5.9

7.5

111*

20

7.5

26*

8

15

116*

20

15

18*

* calculated via regression analysis, first-order model

** calculated via regression analysis, zero-order model

# values excluded due to conditioning of soil community

a) “Following DCD application, soil ammonium concentration was considered a proxy by Irigoyen et al. (2003) and a zero-order model was fitted to these degradation data according to Bronson et al. (1989).”

 

 

Table 2: Data used to plot t½ and soil temperature.

Authors

Temperature in °C

Amount of DCD in kg DCD/ha

Modelled half-life in days

Rajbanshi et al. 1992

10

13

52

20

13

16

30

13

13

10

32

70

20

32

18

30

32

12

Irigoyen et al. 2003a)

10

5.2

> 105

20

5.2

18

30

5.2

7

Di and Cameron (2004)

 

 

8

7.5

111

20

7.5

26

8

15

116

20

15

18

 

The regression analysis revealed following formula t½(T) = 168e-0.084T, accounting for 85 % of the variance with parameter standard errors of ±16 and ± 0.011. See figures 1 and 2.

Conclusions:
The regression analysis revealed following formula t½ (T) = 168e^(-0.084T), accounting for 85 % of the variance (adjusted R²) with parameter standard errors of ±16 and ± 0.011. The analysis of the published data showed a strong dependence of DCD half-lives on the temperature.
Executive summary:

The biodegradation of dicyandiamide (DCD) was assessed in several studies which were used as basis for the present publication. The half-lifes from 16 experiments presented from four different publications were used to derive a formula describing the temperature dependence of the half-life. The following data were used as basis:

Soil no# 1: No data, Amberger 1989.

Soil no# 2: Giessen, Germany: Silt loam, 29 g organic carbon/kg, 2.5 g total nitrogen/kg, pH: 6.1; source: Rajbanshi et al. 1992.

Soil no# 3: Navarra, Spain: Sandy loam, 7.3 g organic carbon/kg, 1.3 g total nitrogen/kg, pH 7.5; source: Irigoyen et al. 2003a.

Soil no# 4: Lincoln, New Zealand: Silt loam, 36.5 g organic carbon/kg, 3.5 g total nitrogen/kg, pH: 5.9; source: Di and Cameron 2004.

 

All tests were conducted under aerobic conditions, and the water content of incubated samples was maintained near the soil’s field capacity. DCD was applied at the rate of 5.2, 7.5, 13, 15 and 32 kg DCD/ha.

The experiment was not conducted in accordance with standardised guidelines or in accordance with GLP.

Details on the test systems of the publications referred to are not presented. Transformation products were not mentioned in the review paper, as well as the incubation time. 

The half-lives of DCD in aerobic soil ranged between 7 and 147 days, depending on the temperature and the amount of DCD added, as well as the type soil. A first-order model was fitted to a plot of t½ and soil temperature (T). The synthesis analysis included 16 measurements from the following studies:

Amberger 1989 (soil #1):

6 °C               No dataon DCD application     t ½ =98 d

           0 °C               No dataon DCD application      t ½ =147 d

           12 °C             No dataon DCD application      t ½ =42 d

 

Rajbanshi et al. 1992 (soil #2):

10°C              13 DCD in kg DCD/ha             t ½ = 52 d

           20°C              13 DCD in kg DCD/ha             t ½ = 16 d

           30°C              13 DCD in kg DCD/ha             t ½ = 13 d

           10°C              32 DCD in kg DCD/ha             t ½ = 70 d

           20°C              32 DCD in kg DCD/ha             t ½ = 18 d

           30°C              32 DCD in kg DCD/ha             t ½ = 12 d

 

Irigoyen et al. 2003 (soil #3)

10°C              5.2 DCD in kg DCD/ha            t ½ = > 105 d

           20°C              5.2 DCD in kg DCD/ha            t ½ = 18 d

           30°C              5.2 DCD in kg DCD/ha            t ½ = 7 d

 

Di and Cameron (2004 soil #4):

           8°C                7.5 DCD in kg DCD/ha            t ½ = 111 d

           20°C              7.5 DCD in kg DCD/ha            t ½ = 26 d

           8°C                15 DCD in kg DCD/ha             t ½ = 116 d

           20°C              15 DCD in kg DCD/ha             t ½ = 18 d

 

The regression analysis revealed following formula t½(T) = 168e-0.084T, accounting for 85 % of the variance with parameter standard errors of ±16 and ± 0.011.

Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009-05-29 to
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: DCD was applied on several plots on farmland
- Short description of test conditions: DCD was applied at 10 kg/ha; Soil samples were collected from the 0–0.1, 0.1–0.2 and 0.2–0.4 m depth layers.
- Parameters analysed / observed:Measurement of DCD concentration
GLP compliance:
not specified
Remarks:
Not mentioned in the published literature.
Test type:
field trial
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
other: Hewitt AE (1998) ‘New Zealand Soil Classification.’ Landcare Research Science Series, No. 1. 2nd edn. (Manaaki Whenua Press: Lincoln, NZ)
Year:
2009
Soil no.:
#1
Soil type:
other: Otorohanga silt loam, a Typic Orthic Allophanic soil; 0-0.1 m depth
% Org. C:
9.3
Bulk density (g/cm³):
700
Soil no.:
#1
Soil type:
other: Otorohanga silt loam, a Typic Orthic Allophanic soil; 0.1 - 0.2 m depth
% Org. C:
6.7
Bulk density (g/cm³):
680
Soil no.:
#1
Soil type:
other: Otorohanga silt loam, a Typic Orthic Allophanic soil; 0.2 - 0.4 m depth
% Org. C:
3.5
Bulk density (g/cm³):
650
Details on soil characteristics:
SOIL COLLECTION AND STORAGE
- Geographic location: Tokanui Dairy Research Farm, located 7 km south of Te Awamutu, New Zealand (38.0◦S, 175.3◦E, 50 m asl)
- Pesticide use history at the collection site: not reported
- Collection procedures: not reported
- Sampling depth (cm): 0 – 0.1 m, 0.1 – 0.2 m, 0.2 – 0.4 m
- Storage conditions: not reported
- Storage length: not reported
- Soil preparation (e.g., 2 mm sieved; air dried etc.): not reported

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm (%): not reported
- Bulk density (g/cm³): 0-0.1 m depth = 700 kg/m³; 0.1 - 0.2 m depth = 680 kg/m³; 0.2 -0.4 m depth = 650 kg/m³
Soil No.:
#1
Initial conc.:
11 kg/ha d.w.
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on experimental conditions:
1. PRELIMINARY EXPERIMENTS:
not reported
2. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): n.a.
- Soil condition: fresh
- Control conditions, if used (present differences from other treatments, i.e., sterile/non-sterile, experimental conditions): n.a.
- No. of replication controls, if used: n.a.
- No. of replication treatments: Five trials with each 6 plots. A trial began with DCD application (10 kg/ha) to six plots, each 5 m by 0.5 m and surrounded by a 0.5 m-wide buffer. Except for one trial beginning 22/4/10, when DCD was applied alone, dairy cattle urine was also applied at an equivalent nitrogen (N) application rate of 700 kg N ha−1 (∼10 L/m²).For each trial, following DCD application, soil in the plots was sampled weekly for the first month and at two week intervals for the second and third months. For the first trial, there were two additional sets of samples taken at monthly intervals.
- Test apparatus (Type/material/volume): field trial: Five trials were conducted during autumn
(1) beginning 29/5/09,
(2 and 3) beginning 22/4/10
(4 and 5) beginning 12/4/11.
- Details of traps for CO2 and organic volatile, if any: no traps
- If no traps were used, is the system closed/open: open
- Identity and concentration of co-solvent: water

Test material application
- Volume of test solution used/treatment: 10 kg DCD/ha
- Application method (e.g. applied on surface, homogeneous mixing etc.): not reported
- Is the co-solvent evaporated: n.a.

Any indication of the test material adsorbing to the walls of the test apparatus: no

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: n.a.
- Continuous darkness: No

Other details, if any:

3. OXYGEN CONDITIONS (delete elements as appropriate)
- Methods used to create the an/aerobic conditions: n.a.
- Evidence that an/aerobic conditions were maintained during the experiment (e.g. redox potential): no

Key result
Soil No.:
#1
DT50:
23 d
Type:
(pseudo-)first order (= half-life)
Temp.:
17.2 °C
Remarks on result:
other: DCD alone
Transformation products:
not measured
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
not measured

Table 2: DCD half-lives (t½), estimated by regression analysis from the data of five field trials as shown in Fig. 1, and percentages of the DCD in sampled soil layers with depths of 0–0.1, 0.1–0.2 and 0.2–0.4 m.

Trial

Year

Treatment

t½[ds]a

days

R–t½[Rs–t½]b

mm

DCD in soil layer

0–0.1 m

%

0.1–0.2 m

%

0.2–0.4 m

%

1

2009

DCD+urine

42 [41]

136 [136]

15

63

22

2

2010

DCD+urine

35 [28]

106 [43]

86

12

2

2

2010

DCD+urine

35 [42]

106 [152]

46

33

21

3

2010

DCD alone

23 [21]

35 [33]

86

11

3

4

2011

DCD+urine

20 [22]

93 [102]

66

30

3

5

2011

DCD+urine

18 [15]

90 [90]

76

23

1

a) ds, days after DCD application when the soil was sampled.

b) R–t½, rainfall during the t½period; Rs–t½, rainfall from DCD application until the soil was sampled.

Table 3: Initial DCD concentrations and corresponding half-lives.

C0 [kg/ha]

t 1/2 [days]

Trial 1

12

42

DCD + urine

Trial 2

11

23

DCD alone

Trial 3

11

35

DCD + urine

Trial 4

9

20

DCD + urine

Trial 5

10

18

DCD + urine

 

Conclusions:
Dicyandiamide (DCD) was applied to pasture plots during autumn in five field trials, with and without dairy cattle urine. DCD concentration results could be fitted to the following first-order exponential degradation model: t1/2 = 54-1.8*T. The experimentally determined half-life for an initial DCD concentration of 11 kg/ha equals 23 days (17.2°C) (Trial 2 DCD application alone).
Executive summary:

The biotransformation of dicyandiamide was studied in a silt loam soil (pH not reported, organic carbon 9.3 % in the first layer, i.e. 0 to 0.1 m depth, 6.7 % in the depth of 0.1 to 0.2 m, and 3.5 % in the depth of 0.2 to 0.4 m) from New Zealand, Otorohanga, for 21 to 42 d under aerobic conditions in the field at ca. 6.7 to 20 ºC. Moisture content was not reported. Dicyandiamide was applied at the rate of 10 kg/ha.  The field trials were undertaken at Tokanui Dairy Research Farm, located 7 km south of Te Awamutu, New Zealand (38.0◦S, 175.3◦E, 50 m asl). The soil’s name is Otorohanga silt loam, a Typic Orthic Allophanic soil according to the New Zealand classification. Volatile compouns were not collected. The soil samples were extracted with water and the DCD residues were analysed by HPLC and each sample’s DCD concentration was calculated on an oven-dry weight basis, having been corrected for bulk density, water content and efficiency of the water extraction process.

 

Transformation products were not assessed.

The following half-lives were determined:

Table 2: DCD half-lives (t½), estimated by regression analysis from the data of five field trials as shown in Fig. 1, and percentages of the DCD in sampled soil layers with depths of 0–0.1, 0.1–0.2 and 0.2–0.4 m.

Trial

Year

Treatment

t½[ds]a

days

DCD in soil layer

0–0.1 m

%

0.1–0.2 m

%

0.2–0.4 m

%

1

2009

DCD+urine

42 [41]

15

63

22

2

2010

DCD+urine

35 [28]

86

12

2

2

2010

DCD+urine

35 [42]

46

33

21

3

2010

DCD alone

23 [21]

86

11

3

4

2011

DCD+urine

20 [22]

66

30

3

5

2011

DCD+urine

18 [15]

76

23

1

a) ds, days after DCD application when the soil was sampled.

 

 

Based on these results following formula was derived: t1/2= 54-1.8*T.

Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
March 2010 to August 2011
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:
- Principle of test:Application of DCD on pasture; remaining DCD analysed after several days.
- Short description of test conditions: field conditions: aerobic, silt loam, day-night cycle.
- Parameters analysed / observed: Remaining DCD in the soil (0-10 cm and 10 -20 cm depth).
A field study was conducted to evaluate the seasonal variations in the biophysical disappearance of DCD in soil, assess the effect of N source on DCD degradation, and determine the major factors controlling seasonal variation.
DCD was applied on two different experimental sites within a farm: a field-plot experiment site (non-grazed) and cattle- grazed site. The non-grazed site was fenced off for a month before the application of treatments to minimise the effect of previous grazing events. DCD was recovered by the water extraction method from soils of the experimental site. Treatments included two levels of DCD alone (10 and 20 kg/ha) applied to non-grazed pasture field plots, and DCD (10kg/ha) applied with urine and with urea fertiliser. DCD (10 kg/ha) was also applied in grazed farmland following grazing. Transformation products or CO2 evolution was not assessed.
GLP compliance:
not specified
Remarks:
Not mentioned in the publishe literature
Test type:
field trial
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
other: Hewitt AE (1998) ‘New Zealand Soil Classification.’ Landcare Research Science Series, No. 1. 2nd edn. (Manaaki Whenua Press: Lincoln, NZ)
Year:
2012
Soil no.:
#1
Soil type:
silt loam
% Clay:
23
% Silt:
68.4
% Sand:
8.5
% Org. C:
3.6
pH:
5.8
CEC:
22.3 other: cmolc/kg
Bulk density (g/cm³):
1.1
Soil no.:
#2
Soil type:
silt loam
% Org. C:
3.2
pH:
5.8
Bulk density (g/cm³):
1.3
Details on soil characteristics:
SOIL COLLECTION AND STORAGE
- Geographic location: Massey University Research Dairy Farm 4, Palmerston North, Manawatu, New Zealand (40°23'40"S,175°36'28"E) in 2010 and 2011. The soil is a poorly drained, Tokomaru silt.
- Pesticide use history at the collection site: not reported
- Collection procedures: At the non-grazing site, 10 intact soil cores (diameter 25 mm) were collected in each plot (2.5 m by 2.5 m) with a soil auger. At the cow-grazing site, 24 intact soil cores (diameter 25 mm) were collected in each plot (600-1000 m2) at soil depths of 0-10 and 10-20 cm. The number of soil cores collected was different at the non-grazing site and cow-grazing site because the plot size at the sites was different. For plant sampling, a 20-cm-diameter ring was randomly located in each plot and all plants inside the rings were cut to 2-3 cm.
- Sampling depth (cm): 0-10 and 10-20 cm.
- Storage conditions: The soils and plant samples were then transferred to the laboratory and processed within 3 h.

- Storage length: no storage
- Soil preparation (e.g., 2 mm sieved; air dried etc.): Field-moist soil samples were sieved through a 4-mm sieve, and subsamples were used to determine soil moisture contents.

PROPERTIES OF THE SOILS (in addition to defined fields)
see Table 1.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on experimental conditions:
1. PRELIMINARY EXPERIMENTS:
A preliminary laboratory experiment was performed to evaluate the DCD recovery rate of the water extraction method that was used to quantify soil DCD in this study.
2. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): field study
- Soil condition:fresh
- Control conditions, if used (present differences from other treatments, i.e., sterile/non-sterile, experimental conditions): no control
- No. of replication controls, if used: n.a.
- No. of replication treatments: 6 to 9
- Test apparatus (Type/material/volume): grazed and non-grazed pasture
- Details of traps for CO2 and organic volatile, if any: not used
- If no traps were used, is the system closed/open: open - field study
- Identity and concentration of co-solvent: deionised water

Test material application - Field-plot experiments
- Volume of test solution used/treatment: 12.5 g DCD in 1 L water; two application rates: 10 and 20 kg/ha
- Application method (e.g. applied on surface, homogeneous mixing etc.): spraying
- Is the co-solvent evaporated: dissolved in water
- Any indication of the test material adsorbing to the walls of the test apparatus: no
- Further details: Two rates of DCD (10 and 20 kg DCD/ha) were applied with no N input in six replicated plots (2.5 m by 2.5 m) on three occasions between early and late spring (August, October, November 2010; Southern Hemisphere). The permanent ryegrass-clover pasture in the treatment plots was cut to a height of 5 cm with a John Deere JX80 mower to mimic a grazing effect. After cutting, a solution of DCD (12.5 g DCD in 1 L water) was evenly sprayed on plots with a hand sprayer. This DCD application technique is similar with the commercial practice used in New Zealand. Soil sampling (soil depth 0-10 cm) for all plots was initially conducted 1 or 2 days after the DCD application and then every 3 days for the first week, then weekly or bi-weekly for the rest of the study period.
Seasonal variation of biophysical disappearance of DCD: from different soil depths (0-10 and 10-20 cm). DCD at 10 kg/ha was applied in six replicated plots (2.5 m by 2.5 m) in March, June, August, and October 2010 and April, June, and August 2011. As described above, the permanent ryegrass- clover pasture in the treatment plots was cut to a height of 5 cm to mimic a grazing effect. Then a solution of DCD (12.5 g DCD in 1 L water) was evenly sprayed on the plots with a hand sprayer. Soil sampling (soil depths 0-10 and 10-20 cm) for all plots was conducted.
At the cattle-grazing farmlet site, experiments were conducted to assess the seasonal differences in biophysical disappearance of DCD by applying 10 kg DCD/ha to nine replicated farmlets (each plot size 600-1000 m2) with a tractormounted Spray unit on six occasions (March, April, and October 2010 and March, April, and June 2011) 2-3 days after cattle grazing (160~300 cows/ha). Soil sampling (soil depth 0-10 cm) on all plots was initially conducted 1 or 2 days after the DCD application and then at weekly or bi-weekly intervals for the rest of the study period.
Experimental conditions (in addition to defined fields)
- Moisture maintenance method: n.a.
- Continuous darkness: No

3. OXYGEN CONDITIONS (delete elements as appropriate)
- Methods used to create the an/aerobic conditions: n.a.
- Evidence that an/aerobic conditions were maintained during the experiment (e.g. redox potential): not reported

4. SUPPLEMENTARY EXPERIMENTS:
Influence of N-input on DCD degradation and DCD persistence on plant canopy

5. SAMPLING DETAILS
- Sampling intervals: Soil sampling (soil depth 0-10 cm) for all plots was initially conducted 1 or 2 days after the DCD application and then every 3 days for the first week, then weekly or bi-weekly for the rest of the study period.
- Sampling method for soil samples:
- Method of collection of CO2 and volatile organic compounds: not conducted
- Sampling intervals/times for: see above
> Sterility check, if sterile controls are used: n.a.
> Moisture content: determined by oven-drying a subsample at 105°C for 24 h
> Other: bulk density was determined by the core method, analysis of total C and N, soils were air-dried at room temperature and sieved (2 mm), and gravimetric moisture contents determined. Total C and N in the soil were measured by combustion in a Leco FP-2000 CNS
> Sample storage before analysis: no
- Other observations, if any: Soil microclimate and long-term climate data collection: On-site instrumentation was used to collect half-hourly averaged values of soil temperature (at 5 cm, a thermistor probe, C8107, Campbell Scientific, USA), soil moisture (at 5 cm depth, time domain reflectometry probes, C5615, Campbell Scientific, USA), air temperature (107-L Temperature Sensor, Campbell Scientific, USA), and precipitation (CS700-L, Campbell Scientific, USA) throughout the study period. Long-term (1971-2010) air and soil (0-10-cm soil depth) temperature and rainfall data in Palmerston North, Manawatu, were obtained from the National Institute of Water and Atmospheric- Research, New Zealand (www.niwa.co.nzjeducation-and-training/schools/resources/climatelearthternp) and New Zealand‘s National Climate Database (http://cliflo.niwa.co.n2/).
Soil No.:
#1
DT50:
6.5 d
Type:
(pseudo-)first order (= half-life)
Temp.:
16.1 °C
Remarks on result:
other: Data from only DCD aplied to non-grazed pasture.
Remarks:
March 2010
Soil No.:
#1
DT50:
12.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
9.3 °C
Remarks on result:
other: Data from only DCD aplied to non-grazed pasture.
Remarks:
June 2010
Soil No.:
#1
DT50:
10 d
Type:
(pseudo-)first order (= half-life)
Temp.:
11.5 °C
Remarks on result:
other: Data from only DCD aplied to non-grazed pasture.
Remarks:
August 2010
Soil No.:
#1
DT50:
9.1 d
Type:
(pseudo-)first order (= half-life)
Temp.:
13.3 °C
Remarks on result:
other: Data from only DCD aplied to non-grazed pasture.
Remarks:
October 2010
Soil No.:
#1
DT50:
12 d
Type:
(pseudo-)first order (= half-life)
Temp.:
13.1 °C
Remarks on result:
other: Data from only DCD aplied to non-grazed pasture.
Remarks:
April 2011
Soil No.:
#1
DT50:
13.8 d
Type:
(pseudo-)first order (= half-life)
Temp.:
10.7 °C
Remarks on result:
other: Data from only DCD aplied to non-grazed pasture.
Remarks:
June 2011
Soil No.:
#1
DT50:
11.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
13.1 °C
Remarks on result:
other: Data from only DCD aplied to non-grazed pasture.
Remarks:
August 2011
Transformation products:
not measured
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
not specified
Details on results:
RESULTS:
TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: No – Field study
- Anomalies or problems encountered (if yes): no

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: not reported
- Range of maximum concentrations in % of the applied amount of DCD at end of study period: not reported

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: not reported
- Range of maximum concentrations in % of the applied amount at end of study period: not reported

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: n.a.

EXTRACTABLE RESIDUES
- % of applied amount at day 0: 66 % for 10 kg/ha applied and 53.5 % for 20 kg/ha applied
- % of applied amount at end of study period: not reported; only half-lives reported

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: not assessed
- % of applied amount at end of study period: not assessed

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: not assessed

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: not assessed

The half-life results from the different experimental designs are presented in the tables 2 to 6.

Table 2: Initial DCD concentrations (kg/ha) and half-life of DCD (days) in soil (0-10 cm depth) after DCD application at 10 and 20 kg/ha. Values are means ± standard error. P values are for significance of difference in initial concentrations or half-life of DCD between DCD application rates (10 v. 20kg/ha). DCD was applied with no nitrogen treatment at a non-grazing site in August (n=6), October (n =6), and November 2010 (n =-6).

Application date

 

Initial DCD concentrations

Half-life of DCD

Application rate

10 kg/ha

20 kg/ha

P value

10 kg/ha

20 kg/ha

P value

August 2010

 

6.6 ± 0.6

10.7 ± 2.0

0.041

10.0 ± 0.9

10.1 ± 1.2

0.941

October 2010

 

4.5 ± 1.1

8.9 ± 1.0

0.005

9.1 ± 1.2

10.0 ± 0.2

0.437

November 2010

 

7.5 ± 0.1

15.4 ± 0.7

0.005

8.2 ± 2.3

8.3 ± 1.8

0.957

 

 

Table 3: Half-life (days, mean ± standard error) of DCD in soil (0-10 cm depth) treated with DCD at 10 kg/ha alone (control) or combined with urea fertiliser (25kg N/ha) or urine (700 kgN/ha) at a non-grazing site in April (n = 6) and June 2010 (n = 6). P values are for significance of difference in half-life of DCD between the different nitrogen treatments. ND, No data.

Application date

DCD only

Urea + DCD

Urine + DCD

P value

April 2010

14.5 ± 1.5

13.7 ± 0.7

11.3 ± 2.4

0.732

June 2010

11.6 ± 1.0

14.0 ± 2.5

 ND

0.520

 

 

Table 4: Initial DCD concentrations (kg/ha) and half-life (days) of DCD in soil at two depths (0-10 and 10-20 cm depth) following DCD application at 10 kg/ha at a non-grazed site in March, June, August, and October 2010 and April, June, and August 2011. Values are means ± standard error. P values are for significance of difference in initial concentrations or half-life of DCD between soil depths (0-10 v. 10-20 cm). ND, No data; pattern of biophysical disappearance of DCD was not fitted with either a linear model or a first-order exponential model.

Application date

 

Initial DCD concentration

Half-life of DCD

Soil depth

0-10 cm

10-20 cm

P

0-10 cm

10-20 cm

P

March 2010

 

4.34 ±

0.6

0.50±

0.2

0.003

6.5 ±

0.5

ND

ND

June 2010

 

3.67 ±

0.5

0.71±

0.1

0.004

12.9 ±

0.9

25.1 ±

2.6

<0.001

August 2010

 

6.01 ±

0.5

0.62 ±

0.2

0.002

10.0 ±

0.9

ND

ND

October 2010

 

2.97 ±

0.4

0.41 ±

0.2

0.001

9.1 ±

1.2

ND

ND

April 2011

 

2.23 ±

0.6

0.2 ±

0.1

0.004

12.0 ±

1.0

ND

ND

June 2011

 

3.89 ±

0.4

0.18 ±

0.03

0.004

13.8 ±

0.9

ND

ND

August 2011

 

4.40 ±

0.4

0.18 ±

0.03

0.001

11.9 ±

1.5

ND

ND

 

Table 5: Half-life of DCD (days) in soil (0-10 cm depth) on the cow grazed farmlets (n=9). DCD at 10 kg/ha was applied in March, April, and October 2010 and March and June 2011. Median values followed by the same letter are not significantly different at p = 0.05 compared to the results from non-grazing sites for the same period.

Application date

Median

Mean

Standard error

Lower 25 %

Upper 25 %

March 2010

6.9b

6.8

0.7

5.0

8.6

April 2010

10.3a

12.3

0.6

9.2

11.2

October 2010

8.4ab

9.6

0.5

6.9

13.1

March 2011

6.0ab

7.4

2.4

5.8

9.3

June 2011

10.8a

11.0

0.7

9.5

12.4

 

Table 6: Half-life of DCD in soil (0-10cm depth) and mean soil temperature and moisture and cumulative rainfall during DCD lifetime (from initial to the time DCD is not detected). Values-are means ± standard error. Soil temperature is the mean of values recorded at 30-min intervals (0-10 cm depth); soil moisture is the mean of determined values from collected soil DCD samples; rainfall is the cumulative value recorded in each period.

Application date

Half-life (days)

Soil temperature (°C)

Soil moisture (%)

Rainfall (mm)

March 2010

6.8 ± 0.7

16.1

19.2

17.0

April 2010

12.3± 0.6

13.5

25.8

20.6

June 2010

12.9 ± 0.9

9.3

34.9

53.9

August 2010

10.1 ± 0.7

11.5

41.1

145.0

October 2010

9.6 ± 0.5

13.3

36.0

42.9

November 2010

8.3 ± 1.3

16.4

23.2

8.9

March 2011

7.4 ± 2.2

16.5

32.0

29.7

April 2011

12.0 ± 1.0

13.1

44.0

156.5

June 2011

11.8± 0.7

10.7

39.4

213.9

August 2011

11.9 ± 1.5

13.1

40.4

156.5

 

Based on the reported results the authors derived the following relationship: Y = aX+b with a = -0.734 ± 0.18), P = 0.004; and b = 20.110 (± 2.5), P<0.0001; R2= 0.66. Using this formula and the monthly average of soil temperature (40-year average) in Palmerston North, the half-life of DCD for each month was estimated (see table 7).

Table 7: Long-term (1971-2010) monthly average soil temperature (0-10 cm depth) and estimated half-life of DCD in a Tokomaru silt-loam soil.

 

Soil temperature (°C)

Half-life od DCD (days)

January

18.3

6.6

February

18.2

6.7

March

16.1

3.3

April

12.7

10.8

May

10.3

12.5

June

7.8

14.4

July

7.0

15.0

August

8.2

14.1

September

10.2

12.6

October

11.8

11.5

November

14.3

9.6

December

16.9

7.7

 

The results of this study show that 4-40% of applied DCD was retained on pasture canopy from <6 days and up to 16 days, depending on pasture height and time of rainfall following DCD application. The half-life of DCD in soil was not affected by either the amount of DCD (10 or 20 kg/ha) or the nature of N applied (synthetic fertiliser and urine) in a poorly drained, New Zealand, dairy-grazed pasture soil. However, half-life of DCD differed with the season and was 7-13 days during March-November. Soil temperature was a major control factor for the variation, and the half-life was longer in lower soil temperature conditions. The monthly half-life of DCD in soil was calculated using the derived relationship with soil temperature. DCD half-life in soil ranged from 6.6 days in January to 15.0 days in July (annual average 12.7± 1.2 days).

Conclusions:
In the present study a liner relation between temperature and half-life of DCD in soil was found. Based on this DCD half-life in soil varied from 6.6 days (January) to 15.0 days (July) with an annual average of 10.4 days.
Half-lifes based on results applying only DCD (without additional N) on non-grazed pasture ranged from 6.5 days (March 2010) to 13. 8 days (June 2011) with an annual average of 10.9 days.
Executive summary:

In the present study the degradation of dicyandiamide (DCD) in soil was assessed. The following different variables were assessed:

-        influence of additional N-source on the degradation

-        influence of season (temperature and humidity)

-        degradation in two different soil depths (0-10 cm and 10-20 cm)

The biotransformation of DCD was studied in a silt-loam soil (pH 5.8, organic carbon 3.60 %) from New Zealand in a field study. DCD was applied at the rate of 10 and 20 kg DCD/ ha soil. The test system consisted of grazed and non-grazed pastures. Samples were analysed at 1 or 2 days after the DCD application and then every 3 days for the first week, then weekly or bi-weekly for the rest of the study period. The soil samples were extracted with deionised water, and the DCD residues were analysed by HPLC-UV. Identification of the transformation products was not conducted.

No significant difference in degradation of the test item was observed between DCD applied solely or in addition with an N-source. As well no significant difference was found for DCD applied either to non- grazed or kettle grazed pastures Initial concentrations at 10-20 cm soil depth were 10-20 % of those at 0-10 cm soil depth, and <10% of DCD applied reached depths below 10 cm. The half-life of DCD could not be determined at 10-20 cm depth, except for the June 2010 application, revealing a significantly slower degradation (half-life = 25.1 d). All data showed a dependence on the temperature of the soil. The authors derived following linear relationship between temperature and all DCD degradation data: Y = aX+b with a = -0.734 ± 0.18), P = 0.004; and b = 20.110 (± 2.5), P<0.0001; R2= 0.66.

Using this formula with the monthly average of soil temperature (40-year average, 0-10 cm depth) in Palmerston North, the half-life of DCD in silt-loam soil for each month was estimated:

 

Soil temperature (°C)

Half-life of DCD (days)

January

18.3

6.6

February

18.2

6.7

March

16.1

3.3

April

12.7

10.8

May

10.3

12.5

June

7.8

14.4

July

7.0

15.0

August

8.2

14.1

September

10.2

12.6

October

11.8

11.5

November

14.3

9.6

December

16.9

7.7

Description of key information

The DT50 of dicyandiamide was derived based on results from several experiments. The geometric mean of data from 8 studies published in Kelliher et al. 2008, Kim et al. 2012, Kelliher et al. 2014 and Balaine et al. 2015 normalised to 20 °C reveal a DT50 of 11.1 days which is considered as key value.

In addition, studies (Hallinger et al. 1990a and b, Amberger and Vilsmeier 1988, 1979, Vilsmeier 1980) are available from which the following conclusions can be drawn:

1) Chemical breakdown of dicyandiamide is catalyzed by metal oxides.

2) Dicyandiamide can be decomposed by soil bacteria.

3) There are at least two different ways of dicyandiamide catabolism. Both seem to be different from the inorganic catalytic dicyandiamide breakdown with metallic oxides. One biological degradation pathway (Mycobacterium smegmatis/ Rhodococcus sp.) leads to cyanourea, urea, and an unidentified substance. Whereas in Pseudomonas sp. guanidine and another unknown product appear during the degradation.

4) Degradation of dicyandiamide in soils depends on soil moisture, temperature and clay content.

5) Main degradation products in soil are guanylurea, guanidine, urea and ammonium.

Key value for chemical safety assessment

Half-life in soil:
11.1 d
at the temperature of:
20 °C

Additional information

In a weight of evidence approach several studies were evaluated. Some studies could not be rated as Klimisch 1 or 2 due to missing documentation. Nevertheless, all presented data is published in scientific magazines, the method applied is reasonable, and the presented data are in consistence with information from literature rated as Klimisch 2 due to a better documentation. All sources were evaluated in a weight of evidence approach enabling a robust and reliable assessment of the biodegradation of dicyandiamide in soil.

The following studies are available presenting DT50 values for dicyandiamide in soil:

Kelliher et al. 2008 present a review of DT50 values from soils of Germany, Spain, and New Zealand. Kim et al. 2012, Kelliher et al. 2014 and Balaine et al. 2015 specifically assess biodegradation of dicyandiamide in soils from New Zealand.

Based on 16 experimentally determined DT50 values reported in the scientific literature, Kelliher et al. 2008 derive the following formula for calculation of the degradation half-live of dicyandiamide in dependence of the temperature:

DT50 (T) = 168e-0.084T; T = temperature in °C

(accounting for 85 % of the variance with parameter standard errors of ±16d and ± 0.011 per day).

 

Dicyandiamide is used as nitrification inhibitor in agricultural applications. According to the report on FOCUS groundwater scenarios in the EU review of active substances (FOUCS 2000), and the Generic Guidance for Tier 1 FOCUS GroundWater Assessments (ESDAC 2014) 20 °C should be chosen as reference temperature: “Where laboratory data have been obtained in line with current EU guidelines (95/36/EC), the reference temperature will be 20°C. Degradation rates obtained at other temperatures should be corrected to this value before averaging (using the procedures described later in this section) or being used directly in model simulations.”

 

Applying the formula presented by Kelliher et al. 2008 to 20 °C a DT50 of 31 days is predicted.

 

In 2014 Kelliher et al. published an updated formula based on additional biodegradation experiments with DCD:           t1/2(T)= 54-1.8*T;

Where             T =temperature in °C.

According to this formula the DT50 of dicyandiamide is predicted to be 18 days at 20 °C.

 

Kim et al. 2012 derived the following formula on basis of their experiments:

Y = aX+b

Where          Y = DT50

                      X = temperature in °C

a = -0.734 (± 0.18)

b = 20.110 (± 2.5)

Applying this formula, a DT50 DCD at 20 °C would equal 5 days.

Experimentally determined DT50 values reported in the above-mentioned literature were evaluated regarding their reliability and relevance, and were subsequently used to calculate a geometric mean DT50 for the biodegradation of dicyandimaide in soil.

Please note: DT50 values that were calculated based on the formulas as presented above were not considered in DT50 calculations for dicyandiamide; only experimentally determined DT50 values were taken into account to calculate a geometric mean. However, the following experimental values were excluded from the overall database:

-        Results from Hauser and Haselwandter (1990) presented in Kelliher et al. 2008, because the test design was not suitable to determine degradation in soil (the derived DT50value was based on an exposure of an isolated bacteria culture to dicyandiamide in a nutrient solution).

-        A value presented in Kelliher et al. 2008 from Irigoyen et al. (2003) which was reported as DT50> 105 at 10 °C was also excluded. Unbound values can not be consulted for evaluation as they do not represent a defined value.

-        Kelliher et al. 2014 report a DT50value of 23 days without giving the referring temperature. Therefore, this value was excluded as well.

The remaining data set of DT50values was normalised to 20 °C according to the formula provided in ECHA guidance document R.7b, p. 222:

DT50ENV= DT50 test*e([Ea/R]*[1/Tenv-1/Ttest])

Where           DT50ENV= half-life in days at 20°C ( 293.15 K )

                       DT50 test= half-life in days at test temperature

Ea = activation energy = 65.4 kj/mol (generic Ea according to EFSA 2007and recommended to be used in the case of missing data by the ECHA guidance document R.7b, 2017, p. 222)

                       Tenv= 20 ° = 293.15 K

                       Ttest= Test temperature

                       R = gas constant = 8.314*10-3kj/(mol*K)

The following table lists the determined DT50 values for each experiment:

Source

Soil type

DT50 in days at test temperature

Test temperature in °C

DT50 in d normalised to 20 °C

Kelliher et al. 2008 (Hauser and Haselwandter 1990)

 

9

25

Excluded due to test design

Kelliher et al. 2008 (Bronson et al. 1989)

 

 

Silt loam

 

 

26

8

8.3

13

15

8.2

8

22

9.6

Kelliher et al. 2008

(citing Amberger 1989)

 

 

No Data

 

 

98

6

25.5

147

0

20.6

42

12

19.8

Kelliher et al. 2008

(citing Rajbanshi et al. 1992)

Silt loam

52

10

20.2

16

20

16.0

13

30

31.5

70

10

27.1

18

20

18.0

12

30

29.1

22

20

22.0

15

30

36.4

Kelliher et al. 2008

(citing Irigoyen et al. 2003)

Sandy loam

>105

10

Excluded due to unbound value

20

18

16.6

30

7

8.6

Kelliher et al. 2008

(citing Di and Cameron 2004)

Silt loam

11

8

3.5

26

20

26.0

116

8

36.9

105

10

40.7

Kim et al. 2012

Silt loam

10

11.5

4.5

9.1

13.3

4.9

8.2

16.4

5.9

10

11.5

4.5

10

13.3

5.3

8.3

16.4

5.9

14.5

13.1

7.6

11.6

9.3

4.2

13.7

13.1

7.2

14

9.3

5.1

11.3

13.1

5.9

6.5

16.1

4.5

12.9

9.3

4.7

10

11.5

4.5

9.1

13.3

4.9

12

13.1

6.3

13.8

10.7

5.7

11.9

13.1

6.2

25.1

9.3

9.1

6.8

16.1

4.7

12.3

13.5

6.7

9.6

13.3

5.1

7.4

16.5

5.4

11

10.7

4.6

Kelliher et al. 2014

Silt loam

23

Not reported

Excluded due to temperature not being reported

Balaine et al. 2015

Silt loam

15.4

22

18.5

16.9

22

20.3

21

22

25.2

27.6

22

33.1

22.4

22

26.9

23.1

22

27.7

24.7

22

29.6

31.5

22

37.8

Geomean

11.1

 

Considering all available DT50 values the geometric mean of 11.1 days at 20 °C is considered as key value and taken forward for risk assessments.

 

In addition, the following studies are available assessing the degradation pathway of dicyandiamide and the factors influencing the degradation of dicyandiamide:

Amberger and Vilsmeier (1979) investigated the breakdown of dicyandiamide (20 mg dicyandiamide-N/100 g soil) under different moisture conditions in quartz sand with metal oxides and in soils. They were able to show that the chemical breakdown of dicyandiamide is catalyzed by metal oxides through adsorption of water and formation of guanylurea. In soils guanylurea was further degraded to ammonium. The dicyandiamide-transformation-rate increased with low humidity, and, in the presence of metal oxides, is dependent on the specific surface area of different metal oxides.

Hallinger et al. (1990a) isolated bacteria from lumber compost and identified them as Mycobacterium smegmatis (isolate No. 16-1) and as Pseudomonas sp. (isolate 11-1 and 18-1). Both genera showed degradation of dicyandiamide with differing degradation products:

-        Mycobacterium smegmatis (isolate No. 16-1): Cyanourea, urea and an unidentified product

-        Pseudomonas sp. (isolate 11-1 and 18-1): Guanidine and an unidentified product.

Similar results are presented in the publication from Hallinger et al. 1990b. Though the isolates are described as Rhodococcus sp. (isolate 16-1) and Pseudomonas sp. (isolate 11-1):

-        Rhodococcus sp. (isolate No. 16-1): Cyanourea, urea and an unidentified product

-        Pseudomonas sp. (isolate 11-1 and 18-1): Guanidine and an unidentified product.

It is assumed that the publications refer to the same experiments, with a different classification of isolate 16-1. The experiments show that the biologic degradation differs from the abiotic breakdown and that at least two different biotic degradation pathways exist.

Vilsmeier (1980) examined the degradation of dicyandiamide in soil in relation to temperature. They applied 20 mg dicyandiamide-N/100 g soil. At temperatures of 10-90 °C dicyandiamide was metabolized to 14-100 % after 14 days. Small amounts of dicyandiamide (0.67 and 1.34 mg dicyandiamide-N/100g soil) were broken down completely within 20-80 days at 8-20 °C. The study demonstrates that dicyandiamide degradation rises with increasing temperature.

Amberger & Vilsmeier (1988) examined leaching of dicyandiamide after mineral fertilizing and slurry manuring and decomposition of dicyandiamide in flooded soils. After mineral feeding, only 0.6-0.9 % of dicyandiamide applied in 5 years was leached. In sediment flooded with water to a height of 10 to 60 cm, dicyandiamide (20 mg/l) was fully degraded within one year in almost all experiments at aerobic conditions while at anaerobic conditions two thirds were decomposed. 

 

References:

ECHA (2017): Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.7b: Endpoint specific guidance. DOI: 10.2823/84188. European Chemicals Agency, 2017.

EFSA (2007): Opinion on a request from EFSA related to the default Q10 values used to describe the temperature effect on transformation rates of pesticides in soil. EFSA Journal 622:1-32.

ESDAC (2014): Generic Guidance for Tier 1 FOCUS Ground Water Assessments (version 2.2).https://esdac.jrc.ec.europa.eu/public_path/projects_data/focus/gw/NewDocs/GenericGuidance2_2.pdf

FOCUS (2000) “FOCUS groundwater scenarios in the EU review of active substances” Report of the FOCUS Groundwater Scenarios Workgroup, EC Document Reference Sanco/321/2000 rev.2, 202pp.