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Toxicity to soil microorganisms

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Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Jan 2014 to 14 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
yes
Remarks:
Soil moisture was adjusted to 39 % of the WHCmax because at 45 % WHCmax the soil was too wet. Soil respiration on day 0 was measured during 10 consecutive hours instead of 12. These deviations had no impact of the integrity of the study.
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
yes
Remarks:
Soil moisture was adjusted to 39 % of the WHCmax because at 45 % WHCmax the soil was too wet. Soil respiration on day 0 was measured during 10 consecutive hours instead of 12. These deviations had no impact of the integrity of the study.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
- Concentrations:
N-transformation: 0.04, 0.16, 0.63, 2.5, 10 mg/kg dry mass tested against untreated control;
C-transformation: 10, 31.6, 100, 316, 1000 mg/kg soil dry mass tested against an untreated control
- Sampling method:
N-transformation: Aqueous soil extracts on day 0 and 28
C-transformation: Withdrawal of soil aliquots on day 0 and 28
- Sample storage conditions before analysis: No storage reported
Vehicle:
yes
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: The soil for the nitrogen transformation test were amended with powdered lucerne-grassgreen meal (C/N ratio between 12/1 and 16/1) at 5 g/kg dry soil and will then be divided into six portions, i.e. five test item treatments and one untreated control.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Appropriate volumes of the test solutions will be mixed thoroughly with the soil portion for the respective treatment to adjust the desired test item concentration in soil

VEHICLE:
- Demineralised water
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2°C in the dark
Moisture:
Soil moisture [% dry weight]: 7.1 (determined according to DIN/ISO 14240-1)
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): C-transformation test - stainless steel containers with lids (volume 1.9 L); N-transformation test - glass jars with lids (volume 290 mL, Weck GmbH, Germany) . A small gap between the lids and the vessels allowed for gas exchange to avoid anaerobic conditions in the vessel and prevented the soil from desiccating.
- Amount of soil: N-transformation test - 320 g dw test soil; C-transformation test - 800 g dw test soil
- No. of replicates per concentration: 4
- No. of replicates per control: 4

SOIL INCUBATION
- Method: The soil was equally distributed to four test vessels which were incubated at 20 ± 2 °C in the dark for 28 days

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Field soil (LUFA standard soil type F2.3) with natural microbial community, provided by Landwirtschaftliche Untersuchungs-and Forschungsanstalt (LUFA) Speyer, Germany
- Geographical reference of sampling site (latitude, longitude): "rechts der Landauer Str.", field number 826/7 at Offenbach, Rhineland-Palatinate, Germany
- Treatments with pesticides or fertilizers: Uncultivated and no fertilisation during the last four years
- Cation exchange capacity (meq/100 g): 10.7 ± 0.8
- Max. water holding capacity [g/100 g]: 35.6 ± 3.0
- Soil moisture [% dry weight]: 7.1 determined according to DIN/ISO 14240-1
- Microbial biomass C [µg Cmic/g soil]: 193; determined according to DIN/IS011465
- Microbial biomass C [% of Corg]:4.6
- Depth of sampling: 20 cm
- Soil type: sandy loam
- Particle size < 0.002 (%): 8.7 ± 1.9
- Particle size 0.002-0.05 (%): 28.2 ± 4.3
- Particle size 0.05-2.0 (%): 63.1 ± 5.0
- pH (0.01 M CaCI2): 6.8 ± 0.1
- Storage (condition, duration): Stored packed in plastic bags at ambient temperature until the start of the test

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- N-transformation: Determintion of nitrate concentrations in aqueous extracts from soil by a photometric cuvette test (2,6-dimethylphenol transformed to 4-nitro-2,6-dimethylphenol) on day 0 and 28
- C-tranformation: Substrate-induced respriation (SIR) measured as CO2 production following amendment of the test soil sample with glucose (4 g/kg soil); CO2 measured using a soil respiration device based on IR gas analysis under ambient air open flow conditions at 500 mL/min

VEHICLE CONTROL PERFORMED: Yes; water

RANGE-FINDING STUDY
- N-transformation: Test concentrations were based on the results of a previously performed non-GLP rangefinding test which indicated possible effects at a test item concentration of 1.0 mg/kg soil
- C-transformation: Test concentrations were defined by the study director in accordance with the sponsor's representative and based on the results of a previously performed non-GLP range-finding test which indicated possible effects at 1 000 mg/kg soil
Nominal and measured concentrations:
- N-transformation: 0.04, 0.16, 0.63, 2.5, 10 mg/kg dry mass tested against untreated control
- C-transformation: 10, 31.6, 100, 316, 1000 mg/kg soil dry mass tested against an untreated control
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
2.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: EC50 calculation not possible because an effect (< 50 % reduction of nitrate formation) was observed in the highest treatment only.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Duration:
28 d
Dose descriptor:
other: LOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Remarks on result:
other: EC50 calculation not possible because an effect (< 50 % reduction of SIR) was observed in the highest treatment only.
Reported statistics and error estimates:
- Normal distribution was assessed using the Kolmogorov-Smirnov test
- Homogeneity of variance was assessed using the Cochran's test
- The Student t-test with Bonferroni-Holm adjustment when variances were found to be homogeneous; in case of inhomogeneous variances, the Welch t-test for inhomogeneous variances with Bonferroni-Holm adjustment was applied.
- Significance level was p < 0.05, two sided.
- All statistical tests were performed with the ToxRat statistical software Version 2.1 0.
Validity criteria fulfilled:
yes
Conclusions:
Validity criteria: The maximum deviation in the carbon transformation test was below the trigger value of 15 % suggested by the guideline.
The authors conclude that the nitrogen transformation rate in soil treated with the test item at a concentration of 10 mg/kg soil dry weight was significantly reduced by 44.3 % on day 28 compared to the control, while no adverse effects were seen on day 0 at any tested concentration. Carbon transformation in soil was significantly reduced on day 0 by 36.1 % and day 28 by 24.1 % in soil treated with 1000 mg/kg soil dry weight. No adverse effects were seen on either with any of the lower test concentrations or on day 0 for any tested concentration. The NOEC for soil microorganisms was 2.5 mg/kg soil dry weight based on nitrogen transformation, and 316 mg/kg soil dry weight based on carbon transformation.
Executive summary:

The effect of cyanoguanidine (dicyandiamide) on the metabolic activity of soil microorganisms was studied according to OECD test guideline 216 (nitrogen transformation) and 217 (carbon transformation). Effect parameters were tested by measuring the nitrate concentration in aqueous soil extracts after 0 and 28 days, and by measuring the substrate-induced respiration rate (SIR) directly from soil aliquots using IR gas analysis.

Effects on nitrogen transformation were tested at nominal concentrations of 0.04, 0.16, 0.63, 2.5, and 10 mg/kg soil dry weight.

Effects on carbon transformation were tested at nominal concentrations of 10, 31.6, 100, 316, and 1000 mg/kg soil dry weight.

Nitrate formation was reduced by 44.4 % at the highest test item concentration of 10mg/kg soil dry weight, whereas no effects were observed at any of the lower test concentrations.

SIR was reduced by 36.1 % at the highest test item concentration of 1000 mg/kg soil dry weight, whereas no effects were observed at any of the lower test concentrations.

Results synopsis

Toxicity to soil microorganisms:

Nitrogen transformation: NOEC: 2.5 mg/kg soil dw

Carbon transformation: NOEC: 316 mg/kg soil dw

This toxicity study is classified as acceptable and satisfies the guideline requirement for a study on inhibitory effects to soil microorganisms.

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reason / purpose:
reference to same 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 were able to break down DCD rapidly. DCD metabolism was further followed by thin-layer chromotography (TLC) on silica gel. DCD did not affect microbial growth and showed no toxicity towards microorganisms.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (inoculum):
other: Rhodococcus, Pseudomonas
Total exposure duration:
3 d
Test temperature:
27 °C
Moisture:
Not applicable > experiment in solution

The degradation of 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 in DCD-supplied bacteria. Pseudomonas sp. behaved similarly: Concomitant 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 were observed after 3 days of culture which never appeared in the controls. Pseudomonas sp. also metabolized DCD in 3 days, forming two metabolites.

Apparently there are at least two different pathways for 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., chromatographic analysis revealed a substance together with guanidine and a further, unknown product. Rhodococcus and Pseudomonas seem to be incapable of metabolizing urea.

In contrast to the DCD degradation by 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.

Validity criteria fulfilled:
not applicable
Conclusions:
As DCD showed no toxic effects towards the cultured bacteria and could be rapidly degraded.
Executive summary:

In this experiment it could be shown that apart form abiotic degradation dicyandiamide can also be degraded biologically. This has been confirmed with bacteria isolated from soils.

To further characterize the catabolism of dicyandiamide, different bacteria have been isolated from dicyandiamide-treated composts 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 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 of Rhodococcus was very rapid: 200 ug 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 in the dicyandiamide-supplied bacteria. Pseudomonas sp. behaved similarly: Concomitant 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 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., Hallinger et al. (1990) found a substance together with guanidine on the chromatrogram and a further, unknown product. It is notable that Rhodococcuss and Pseudomonas seem to be incapable of metabolizing urea (unpublished observations). In contrast to the dicyandiamide degradation by 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. Urea was never detected as a degradation product. The dicyandiamide-degrading principle is heat-labile, as could be shown by boiling for 30 s.

As DCD could be rapidly degraded by these bacteria it is concluded that the substance does not exert any not toxic effects on these bacteria.

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
This study meets generally accepted scientific principles, is well documented and acceptable for assessment. The purity of the test substance is not reported, but this study has been peer reviewed prior to publication and has been conducted according to scientific standards. Therefore, it can be assumed that the test substance used in this test was of adequate purity to allow assessment of effects. Moreover, the result of this study (DCD is not toxic to microorganisms) is supported by the results by the study "461-58-5_DCD_Bacterial_Degradation_1990.pdf".
Qualifier:
no guideline followed
Principles of method if other than guideline:
Ammonium-oxidizing bacteria (Nitrosomonas sp.) were exposed to 100 mg/l dicyandiamide as a dilute aqueous solution for 48 h at 4 °C to allow for any bactericidal action to take effect. After that, the cultures were incubated for 28 days at 28°C. After these four weeks cultures were tested for ammonium oxidation (change in pH, determination of nitrite and nitrate).
GLP compliance:
not specified
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on preparation and application of test substrate:
Dicyandiamide was applied as a dilute aqueous solution to bacteria in a culture medium.
Test organisms (inoculum):
other: Nitrosomonas sp. (soil)
Total exposure duration:
30 d
Remarks:
two days of preincubation at 4°C to allow time for any bactericidal action to take effect, but not to permit any appreciable growth of surviving bacteria; after that incubation for 28 days at 28°C
Test temperature:
Preincubation: 4 °C
Main test: 28 °C
Moisture:
Not applicable
Details on test conditions:
Ammonium-oxidizing bacteria (Nitrosomonas sp.) were obtained from soil from Woburn Experimental Farm and grown in a 100-ml batch culture as described by Rodgers et al. (1980). When bacteria in the enrichment culture were in the early stationary phase of growth the culture medium was divided into two portions, one untreated and the other given the requisite dose of the inhibitor. Dicyandiamide was applied as a dilute aqueous solution.
The untreated portion of the culture was serially diluted in inorganic medium containing phenol red indicator in flasks which were then plugged with cotton wool, capped with aluminium foil, and gently rotated in an orbital incubator at 24 °C in the dark. Serial dilutions were prepared at a dilution ratio of 10 with six levels (1:102 to 1:107) and five replicates at each level.
The treated portion was kept in a plugged and capped flask at 4 °C for 48 h before similar serial dilution. This period was chosen to allow time for any bactericidal action to take effect, but not to permit any significant growth of surviving bacteria.
The serially diluted cultures were examined (after incubating for 28 days at 28 °C) for a colour change from red to yellow indicating a decrease in pH from approximately 8 to approximately 6 due to ammonium oxidation. The diluted cultures were also tested with nitrite and nitrate test papers (E. Merck, Darmstadt) to confirm ammonium oxidation and to ensure that any nitrite produced had not been further oxidized to nitrate due to contamination by nitrite-oxidizing bacteria. The “most probable number” of ammonium oxidizers present in the early stationary phase enrichment culture, before and after the 48 h treatment, was calculated from the number of replicates at each dilution level that were acidified after 4 weeks.
Nominal and measured concentrations:
100mg/l (nominal)
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
other: inhibition of nitrification
Effect conc.:
100 other: mg/l
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: pH reduction due to ammonium oxidation
Remarks on result:
other: complete inhibition
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
> 100 other: mg/l
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: bactericidal effects
Remarks on result:
other: no bactericidal effects observed at 100 mg/l
Details on results:
In the experiment with Nitrosomonas sp. the number of viable ammonium oxidizers was not significantly affected by dicyandiamide at 100 mg/l although nitrification was completely inhibited. Increasing the time of exposure to the inhibitor beyond 48 h did not result in a bactericidal effect either.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
Testing for significant differences between the number of viable ammonium oxidizers in treated and untreated cultures.

Table 1: Effect of dicyandiamide on ammonium-oxidizing bacteria in culture medium

 

 

After 48h treatment with inhibitor

Untreated

 

Inhibitor

mg inhibitor/ L medium

Mean no.

Lower limit

Upper limit

Mean no.

Lower limit

Upper limit

Significant bactericidal action

Dicyandiamide

100

3.3 x 104

1.0 x 104

1.0 x 105

3.3 x 104

1.0 x 104

1.0 x 105

No

 

Validity criteria fulfilled:
not applicable
Remarks:
no specific guideline followed
Conclusions:
Dicyandiamide is not bactericidal to Neutrosomonas sp. but inhibits nitrifcation (bacteriostatic effect).
Executive summary:

Dicyandiamide was investigated in an attempt to establish whether it actually kills ammonium-oxidizing bacteria (bactericidal action) or whether bacteria remain viable but temporarily incapable of nitrification (bacteriostatic action). In laboratory experiments with nitrifying cultures, nitrification was completely inhibited, but the numbers of ammonium-oxidizing bacteria were not significantly affected in a 48-h treatment with dicyandiamide applied at the rate of 100 mg/L culture medium.

Description of key information

A study on the inhibitory effects of cyanoguanidine (dicyandiamide) to the metabolic performance of soil microorganisms was conducted. The method followed the OECD test guidelines 216 and 217 (Soil microorganisms: nitrogen transformation test/carbon

transformation test). Decrease of metabolic activity was observed in both studies at the highest test concentration, respectively.

The NOEC for nitrogen turnover was 2.5 mg/kg soil dw, and the NOEC for carbon transformation was 316 mg/kg soil dw.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
2.5 mg/kg soil dw

Additional information

Dicyandiamide is known as a nitrification inhibitor since decades (e.g. Rodgers et al., 1982). Inhibition of nitrification is a desired property of dicyandiamide that is uitilised in agricultural applications. The substance is approved as an additive to fertilisers according to Regulation (EU) No 2003/2003 (consolidated version of 4th April 2013), specifying a permitted range of 2.25–4.5 % (w/w) in terms of total nitrogen present in the fertiliser. Inhibition of nitrification results in a slowdown of the transformation of poorly mobile forms of nitrogen (ammonium) into a highly mobile form (nitrate). In effect, nitrogen from dicyandiamide-amended fertilisers is available to plants over longer period, and it is displaced to deeper soil layers and groundwater at a significantly lower rate. Accordingly, the inhibiting action of dicyandiamide on nitrification can be considered to have positive effects on the protection of groundwater from nitrate leaching. Furthermore, it has been shown by several studies (IUCLID section 5.2.3) that dicyandiamide is relatively quickly degraded in soil, indicating that any negative effects on the soil microflora are only mild. In conclusion, the result from the test on the effects on nitrogen turnover (OECD 216) are considered as an adverse effect relevant for the chemical safety assessment regarding the risk assessment of industrial chemicals und REACH as a release of dicyandiamide to the soil from industrial applications is not intended. However, in agricultural applications the desired property of nitrification inhibition is not regarded as an adverse effect but rather an intended effect. Instead, the effect concentrations identified in the carbon transformation test are considered to be more critical.