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Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data, publication
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
This study meets generally accepted scientific standards, is well documented and acceptable for assessment. The purity of 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 substsance 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 be the results by the study "461-58-5_DCD_Toxicity_Cell_Multiplication_Bacteria_1988.pdf"
Reason / purpose for cross-reference:
reference to same study
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 48h at 4 °C to allow time 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 test solutions:
Dicyandiamide was applied as a dilute aqueous solution to bacteria in a culture medium.
Test organisms (species):
Nitrosomonas sp.
Details on inoculum:
Ammonium-oxidizing bacteria (Nitrosomonas sp.) were obatained from soil from Woburn Experimental Farm and grown in a 100-ml batch culture as described by Rodgers et al. ( "Recovery of nitrifiers popuplations from inhibition by nitrapyrin or carbon disulphide", Zentralbl. Bakteriol. Parasitkd. Infektionskr. Hyg. Abt. 2, 135: 477-483, 1980)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 d
Remarks on exposure duration:
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
Post exposure observation period:
no post exposure observation period
Hardness:
not applicable
Test temperature:
not applicable
pH:
not applicable
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
100 mg/l (nominal)
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 (1:10^2 to 1:10^7) and five replicates at each level.
The treated portion was kept in a plugged and capped flask at 4°C for 48h before similar serial dilution. This period was chosen to allow time for any bactericidal action to take effect, but not to permit any appreciable 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 fall 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 by contaminating nitrite-oxidizing bacteria. The “most probable number” of ammonium oxidizers present in the early stationary phase enrichment culture, before and after the 48h treatment, was calculated from the number of replicates at each dilution level that were acidified after 4 weeks.
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
other: inhibition of nitrification
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reduction in pH due to ammonium oxidation
Remarks on result:
other: complete inhibition
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
> 100 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 100mg/l although nitrification was completely inhibited. Increasing the time of exposure to inhibitor beyond 48h did not evolve a bactericidal effect.
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 organisms 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:
At 100 mg/l Dicyandiamide is not bactericidal towards Neutrosomonas sp. but inhibits nitrifcation (bacteriostatic effects).
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 numbers of ammoniumoxidizing bacteria were not significantly affected by a 48-h treatment with dicyandiamide applied at the rate of 100 mg inhibitor/L culture medium.

Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May - June 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, well documented. Recommended study in context of classification into water hazard classes
Remarks:
GLP, well documented. Recommended study in context of classification into water hazard classes
Principles of method if other than guideline:
The multiplication of bacterial cells of Pseudomonas putida is inhibited by dissolved toxic water ingredients. After a certain period, the increase in the number of cells in a test culture free from toxic influence and with a defined standardized quantity of nutrients will exceed that observed in a test culture containing dissolved toxic substances and kept under identical conditions.
The concentration of bacterial suspension is measured turbidimetrically; it is expressed as the extinction of the primary light of monochromatic radiation at 436 nm for a layer thickness of 10 mm. The concentration at which the inhibitory action of a test substance starts, will be present in that step of a dilution series of the test substance having an extinction value at the end of the test period that is ≥ 10 % below the mean extinction value of the control cultures. This toxicity threshold (TT) of the test substance for Pseudomonas putida will be determined graphically.
GLP compliance:
yes
Specific details on test material used for the study:
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 209-211 °C
- Solubility in water: 32 g/l (20°C)
- specific gravaity: 1.4 g/cm³
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- A test substance solution of 32000 mg Dicyandiamide/l in sterile Mill-Q water (Millipore Corp., Bedford, mass., USA) was prepared.
- The test substance solution was diluted 1.25* in sterile Milli-Q water.
- The pH of the test substance was adjusted to 7.1 using an appropriate HCl solution to ensure minimal increase of volume.

STOCK SOLUTION I
- 20.000 g D(+)-glucose (for biochemical and microbiological purposes)
- 4.240 g sodium nitrate (NaNO3), A.R.
- 2.400 g dipotassium hydrogen phosphate (K2HPO4)
- 1.200 g potassium dihydrogen phosphate (KH2PO4), A.R.
- 30 ml trace elements solution
- Glucose and nutrient salts were dissolved each separately in 500 ml Mill-Q water, sterilized in a steam sterilizer and united when cooled

STOCK SOLUTION II
- 0.200 g ferrous sulphate, FeSO4*7H2O, A.R.
- 4.000 g magnesium sulphate, MgSO4*7H2O, A.R.
- Both nutrients were dissolved in Milli-Q water and sterilized in a steam sterilizer
Test organisms (species):
Pseudomonas putida
Details on inoculum:
STOCK CULTURES OF PSEUDOMONAS PUTIDA
- stock cultures of Pseudomonas putida were obtained from RIVM, Bilthoven, The Netherlands
- Stock cultures were kept on the nutrient used for stock and preliminary cultures in agar plant tubes
- new stock cultures were started at intervals of 1 week
- the inoculated stock cultures were incubated at 25 °C

PRELIMINARY CULTURES OF PSEUDOMONAS PUTIDA
- usind aseptic techniques, small amounts of bacteria from a 7-day old stock culture of Pseudomonas putida were inoculated in fluid nutrient medium in Erlenmeyer flask
- the preliminary cultures were incubated at 25 °C for 18+/-2 hours
- subsequently the extinction of the monochromatic radiation at 436 nm for a 10 mm layer of the bacterial suspension was determined by photoeletric measurement
- on the basis of the values measured, the final turbidity value of the bacterial suspension was adjusted by means of sterile saline in such a way that the extinction value for a sample that ahs been subject to onward dilution 1 + 9 with ssaline corresponds with the extinction value of a Formazin standard suspension TU/F/436nm=10.
- these preliminary cultures of bacterial suspensions were used for inoculation of the test flasks
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
18
Post exposure observation period:
Not applicable
Hardness:
No data
Test temperature:
25 °C
pH:
7.1
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations:
2^0, 2^2, 2^4, 2^6, 2^7, 2^8, 2^10, 2^11, 2^12, 2^13, 2^14, 2^15 and 2^16 of Dicyandiamide (mg/l)
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 ml Erlenmeyer flasks toppered with aluminium caps
- No. of vessels per concentration (replicates): 4 parallele dilution series


TEST PROCEDURE
- Each of the dilutions contained 1 part v/v of the test substance solution in 2^0 to 2^11 parts v/v mixture.
- The dilution series were prepared as follows:
* the first flask of each series contained 160 ml of the test substance solution, from this flask the subsequent dilution steps at a constant
dilution ratio were prepared by consistently mixing 80 ml of preliminary test substance dilution and 80 ml sterile Milli-Q water
* Consequently, each flask contained 80 ml of the test substance solution at the start.
* Each flask of the three inoculated dilution series (I, II, III) was made up to a finla volume of 100 ml by adding 5 ml of a stock solution I (see "details
on test solutions"), 5 ml of a stock solution II and 10 ml of the prepared bacterial suspension from the preliminary culture having a known adjusted
extinction value
* Following inoculation, the extinction value of 436 nm will correspond to the extinction value of the Formazin standard suspension
TU/F/436nm=10.
* The flasks of the dilution series that were not inoculated (series t), were made up to 100 ml by adding 5ml of stock solution I, 5 ml of stock
solution II and 10 ml saline.
* Ten control culture flasks (series B) with 80 ml sterile Milli-Q water, 5 ml stock solution I, 5 ml stock solution II and 10 ml prepared bacterial
solution were also included.
- Both inoculated and non-inoculated dilutiuon sereis as well as the control culture flasks were left at at 25 °C for 18 +/- 2 hours
- After termination of the test period the cell suspensions were homogenized at the extinction of the monochromatic radiation at 436 nm in a 10-mm
layer in the four dilution series and the control flasks was measured.
- If after termination of the test period, colouration or turbidity occurred in the dilution series for chemical-physical reasons, the analogous steps of dilution of the non-inoculated series will be used as phootometric blank values for turbidity of the inoculated dilution series.


OTHER TEST CONDITIONS
- Adjustment of pH: pH adjusted to pH 7.1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
extinction values


RANGE FINDING EXPERIMENT
- concentrations: 2^0, 2^2, 2^4, 2^6, 2^8, 2^10 mg/l Dicyandiamide
Reference substance (positive control):
yes
Remarks:
Methanol (Merck no. 6008, The Netherlands) > known TT value
Duration:
18 h
Dose descriptor:
other: TT
Effect conc.:
130.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell multiplication
Duration:
18 h
Dose descriptor:
other: Bewertungszahl
Effect conc.:
3.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell multiplication
Duration:
18 h
Dose descriptor:
EC10
Effect conc.:
126.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell multiplication
Remarks on result:
other: value calculated from Bewertungszahl > Bewertungszahl = -log(EC10/1000000)
Details on results:
RANGE FINDING EXPERIMENT
- no toxicity threshod value could be determined
- therefore, the final experiment was carried out with onward dilutions

MAIN EXPERIMENT
- none of the concentrations in the dilution series of Dicyandiamide showed colouration or turbidity; therefore, no correction for the extinction values of the dilution series was made
- the TT value of Dicyandiamide was 130.6 mg/l
- the "Bewertungszahl" (assessment figure or value for bacteriotoxicity) is therefor 3.9 (Bewertung wassergefährdender Stoffe, 1979)
Results with reference substance (positive control):
- The TT value for methanol determined for Pseudomonas putida was 20348 mg/l.
- With respect to the known literature TT value of methanol (6600 mg/l) and considering the existing differences in definitions for the TT value in the literature concerned, it can be concluded that the test conditions were optimal and the results obtained are valid.
Reported statistics and error estimates:
see table 1

Table 1: Extinction values of the inoculated dilution series I, II, III, the control flask B and the reference dilution series R

EXTINCTION 436 nm

 

 

Methanol

 

Dicyandiamide

Mean

 

B

mg/l

R

mg/l

I

II

III

I, II, III

 

0.524

4937.5

0.541

0.4

0.526

0.520

0.531

0.526

 

0.520

9875.0

0.514

0.8

0.525

0.506

0.522

0.518

 

0.477

19750.0

0.452

1.6

0.506

0.510

0.513

0.510

 

0.501

39500.0

0.175

3.1

0.493

0.515

0.514

0.507

 

0.509

79000.0

0.065

6.3

0.531

0.530

0.532

0.531

 

0.496

 

 

12.5

0.520

0.540

0.515

0.525

 

0.475

 

 

25.0

0.492

0.497

0.486

0.492

 

0.486

 

 

100.0

0.457

0.467

0.468

0.464

 

0.460

 

 

200.0

0.428

0.386

0.378

0.397

 

0.481

 

 

400.0

0.296

0.289

0.285

0.290

 

 

 

 

1600.0

0.165

0.155

0.153

0.158

 

 

 

 

6400.0

0.133

0.135

0.141

0.136

MEAN

 

 

 

25600.0

0.061

0.061

0.058

0.060

 

0.493

 

 

 

 

 

 

 

 

90%-line

                 0.444

Methanol

TT value

20348 mg/l

Dicyandiamide

TT value

130.6 mg/l

 

Validity criteria fulfilled:
yes
Conclusions:
The toxicity threshold value for dicyandiamide is 130.6 mg/l. The "Bewertungszahl" is therefore 3.9.
Therefore, it can be concluded that Dicyandiamide is not toxic towards bacteria.
Executive summary:

A "Acute bacteria Cell Multiplication Inhibition Test" was conducted to determine the acute toxicity of Dicyandiamide on the cell multiplication of a pure culture of Pseudomonas putida bacteria. Pseudomonas putida cells were exposed to Dicyandiamide in concentrations of 20, 22, 24, 26, 27, 28, 210, 211, 212, 213, 214, 215and 216mg/l (0.4 to 25600 mg Dicyandiamide/l). 

Based on the solubility of Dicyandiamide in water, a toxicity threshold value of Dicyandiamide of 130.6 mg/l for Pseudomonas putida could be determined. The “Bewertungszahl” (assessment figure or value for bacteriotoxicity) is therefore 3.9 (Bewertung wassergefährdender Stoffe, 1979).

The EC10(18h), calculated from the Bewertungszahl, is 126.9 mg/l.

Description of key information

In accordance with REACH Annex IX column 2, a test for activated sludge respiration inhibition may be replaced by a nitrification inhibition test if available data show that the substance is likely to be an inhibitor of microbial growth or function, in particular

nitrifying bacteria. According to Umweltbundesamt (UBA 1993) and Reynolds et al. (1987) a test on inhibition of nitrification is more sensitive than a test on growth inhibition with Pseudomonas putida (see ECHA Guidance on information requirements - Chapter R.7N, p. 146).
In a laboratory experiment (461-58-5_DCD_Bacteriostatic _action_1982.pdf) Ammonium-oxidizing bacteria (Nitrosomonas sp.) were exposed to 100 mg/l Dicyandiamide as a dilute aqueous solution for 48h at 4 °C to allow time 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). Nitrification was inhibited but Dicyandiamide had no influence on cell numbers.
In the second study, a “Acute bacteria Cell Multiplication Inhibition Test” was conducted to determine the acute toxicity of Dicyandiamide on the cell multiplication of a pure culture of Pseudomonas putida bacteria. Pseudomonas putida cells were exposed to Dicyandiamide in concentrations of 2^0, 2^2, 2^4, 2^6, 2^7, 2^8, 2^10, 2^11, 2^12, 2^13, 2^14, 2^15 and 2^16 mg/l (0.4 to 25600 mg Dicyandiamide/l) for 18 +/- 2 hours. The EC10 (18h), calculated from the Bewertungszahl determined in this study, is 126.9 mg/l.

Key value for chemical safety assessment

Additional information

In the study " 461-58-5_DCD_Bacteriostatic _action_1982.pdf" 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 this laboratory experiments with nitrifying cultures, nitrification was completely inhibited, but numbers of ammonium oxidizing bacteria were not significantly affected by a 48-h treatment with dicyandiamide applied at the rate of 100 mg inhibitor/L culture medium.

Therefore, it can be concluded that Dicyandiamide is not toxic towards bacteria.

 

These results are supported by the results of the second study ("461-58-5_DCD_Toxicity_Cell_Multiplication_Bacteria_1988.pdf").

Based on the solubility of Dicyandiamide in water, a toxicity threshold value of Dicyandiamide of 130.6 mg/l for Pseudomonas putida could be determined. The “Bewertungszahl” (assessment figure or value for bacteriotoxicity) is therefore 3.9 (Bewertung wassergefährdender Stoffe, 1979). The EC10(18h), calculated from the Bewertungszahl, is 126.9 mg/l.