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EC number: 207-312-8 | CAS number: 461-58-5
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.
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.
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.
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.
Table 1: Effect of dicyandiamide on ammonium-oxidizing bacteria in culture medium
After 48h treatment with inhibitor
mg inhibitor/ L medium
Significant bactericidal action
3.3 x 104
1.0 x 104
1.0 x 105
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.
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.
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.
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