Calcium cyanamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-17 to 2010-10-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Regulation (EC) No 761/2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 23 March 2006

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for chemical analysis were taken of the blank (containing test substance, but no algae) and of one replicate of each of the test and control cultures at the start and at the end of the test and were sent to the Test Site for analysis of the test substance content. For stabilization, the samples were be deep frozen immediately after sampling and stored at -18°C. The transport of the samples to the Test Site was on dry ice. Since there is no analytical method available for the detection of the complete test substance, the cyanamide fraction of the test substance was determined analytically.
"CALCIUM CYANAMIDE contains 36.4 % cyanamide.

Vehicle:

no

Details on test solutions:

The test substance has impurities and is of limited solubility in aqueous media. Therefore the algae were exposed to separate preparations at different loading rates of water-accommodated fractions (WAFs). According to the recommendation of the principle investigator the respective amount of test substance was added to the nutrient medium and stirred for about one hour in the dark.
The nutrient medium consisted of sterile deionised water and sterile concentrated nutrient medium 9+1. The conductivity of the deionised water used was below 5 μScm-1 (determined with a conductivity meter CONMET 1, HANNA Instruments, Germany). After stirring for one hour not dissolved particles were allowed to precipitate and the surplus was filtrated (0.45 μm, Minisart, Sartorius) to obtain clear solutions which were used for the test.
One negative control culture (reference substance: nutrient medium) was set up concurrently. Additionally, one blank containing the highest test substance loading rate used, but no algae, was incubated under the same culture conditions for analysis of the test substance concentration and comparison with the algae cultures. The test was performed without adjustment of the pH of the test substance cultures.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) ATCC (American Type Culture Collection) 22662, obtained from LGC Promochem GmbH, Mercatorstraße 51, 46485 Wesel A Rhein, Germany.
Stock cultures, initially cultivated from deep frozen algae, are continuously maintained in 250 mL conical flasks (Erlenmeyer), each containing 100 mL nutrient medium, at a temperature in the range of 21 to 24 (± 2) °C under permanent light with an intensity between 4400 and 8800 lux. In about weekly intervals 1 mL of the stock culture is diluted 100-fold with nutrient medium for precultivation and incubation is continued. Precultures were prepared by incubating a new stock culture for four days. At the start of the experiment the cell density was determined and adjusted to about 105 algae/mL by diluting with nutrient medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

no data

Test temperature:

21 - 24 (± 2) °C

pH:

The pH was between 7.8 and 11.3 at the start of the incubation in the test cultures and it was 7.8 in the control cultures. After 72 hours of incubation the pH was between 8.0 and 10.0 in the test cultures and it was 9.8 in the control cultures.

The pH in the control culture changed by 2.0 units, i.e. by more than the 1.5 units recommended by the EC directive. As the control culture showed adequate growth, the increase in pH did not adversely affected algal growth or the outcome of the experiment, therefore the change in pH was tolerated.

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal: 0; 0.74; 1.65; 3.27; 6.64; 13.73; 25.88; 49.95; 104.40; 200.25 mg/L
Measured: 0; 0.79; 1.53; 2.53; 6.12; 13.64; 21.37; 28.08; 80.81; 179.63 mg/L
Beside one outlier (nominal 49.95 mg/L), the measured cyanamide concentrations were stable within ± 20 % of the nominal test substance loading rates throughout the duration of the test based on mean values. Some measured values were slightly below 80 % of the nominal loading rates (e.g. nominal 100.40 mg/L). Nevertheless, taking all values into account, it is reasonable that nominal values are used for calculation of the effective loading rates. The nominal value of 49.95 mg/L is regarded as outlier most likely due to inconsistencies during sampling or analytical measurement. Thus, it is scientifically justified to also include this value in the EL50 calculation based on nominal loading rates.

Details on test conditions:

At the onset of the experiment the cell concentration in the preculture was determined and the inoculum was prepared by diluting an appropriate volume of the preculture with nutrient medium to give a calculated cell concentration of 105 cells/mL. All test cultures and the blank were set up in 250 mL conical glass flasks (Erlenmeyer). The total culture volume was 100 mL in each case. Each test substance culture consisted of 10 mL inoculum (105 algae/mL) and of 90 mL of the respective test substance preparation. The blank without algae contained 90 mL of the test substance preparation containing the highest loading rate and 10 mL of nutrient medium. The negative control cultures contained 10 mL of inoculum and 90 mL of nutrient medium.
All cultures and the blank were incubated at 21 - 24 (± 2) °C for three days in permanent light with an intensity between 4400 and 8800 lux (cool white light) while shaking. Three replicate cultures were incubated for each test substance concentration and six replicates for the control.

Determination of the cell density of the algae, visual observations
Cell density acts as the surrogate parameter for algal biomass which is defined as the dry weight per volume (mg algae/L test solution). According to the EC regulation 761/2009 C.3 the conversion factor between cell density and biomass is 2-3 x 10-8 mg/cell. At incubation times of 24, 48 and 72 hours aliquots were taken from each replicate and the cell count of the algae was determined by a Casy Cell Counter, Model DT, Schärfe System GmbH, Germany (pore size 60 μm). To achieve total cell counts of less than 50000, appropriate amounts of the cell suspensions were filled up to 10 mL of isotonic solution (Casyton, Schärfe System). Visual observations on the appearance of the algal cultures were made each time the cell densities were determined.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

other: NOAEL

Effect conc.:

ca. 13.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: NOAEL

Effect conc.:

ca. 6.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 27.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 16.98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Growth inhibition
- Based on the yield and compared to the negative controls: In all loading rates tested, the influence on algal growth ranged from 100.5 % inhibition to 23.2 % enhancement.
- Based on the average growth rates and compared to the negative controls: In all loading rates tested, the influence on algal growth ranged from 100.0 % inhibition to 3.9 % enhancement.

Visual and microscopic observations
No change in the colour of the media and no precipitation of test substance was noted during the incubation period. Microscopic evaluation of the inoculum at the start of the test and of the algae at the end of the test revealed normal appearance of the algae.

Results with reference substance (positive control):

The last reference test with K2Cr2O7 was conducted from the 21st to the 24th of June 2010 with Pseudokirchneriella subcapitata to evaluate the reliability of the test conditions. The 72 hour EC50 for growth rate and yield were 1.32 and 0.62 mg K2Cr2O7/L, respectively. These results establish the reliability of the test procedures for this kind of study type.

Reported statistics and error estimates:

The number of algae/mL is calculated for all individual samples from the cell counts measured at the start of the exposure as well as 24, 48 and 72 hours thereafter.
The mean growth rate of the test substance culture is expressed as percent of the mean growth rate of the control and from this the percent inhibition is calculated. The logarithms of the substance loading rates are then plotted against the corresponding percent inhibition. The ErL50 for the 72 h incubation period is taken from the intercept of this curve with the parallel drawn to the abscissa at 50 % inhibition.
The mean yield of the test cultures is expressed as percentage of the mean yield of the control cultures and from this the percentage inhibition in yield is calculated. The logarithms of the substance loading rates are then plotted against the corresponding percent inhibition. The EL50 for the 72 h incubation period is taken from the intercept of this curve with the parallel drawn to the abscissa at 50 % inhibition.
Based on the yield as well as on the average growth rates two "lowest observed effective loading rates" (LOELs) are calculated by comparison of the data of the three replicates of each test substance culture with the negative control (analysis of variance, followed by the Scheffé-test, P = 0.05). The "no observed effect loading rates " (NOELs) are then derived from these results (highest concentration with no statistically significant difference to the control).

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (72 h) of calcium cyanamide, technical grade (Kalkstickstoff) in algae (Pseudokirchneriella subcapitata) was determined to be 27.54 mg/L based on nominal concentrations (growth rate). The NOEC was determined to be 13.73 mg/L based on growth rate.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to calcium cyanamide, technical grade (Kalkstickstoff) at nominal concentrations of 0; 0.74; 1.65; 3.27; 6.64; 13.73; 25.88; 49.95; 104.40; 200.25 mg/L under static conditions in accordance with EC regulation 761/2009 Part C.3 and the OECD-Guideline 201. The test substance has impurities and is of limited solubility in aqueous media. Therefore the algae were exposed to separate preparations at different loading rates of water accommodated fractions (WAFs).

The NOEC and EC50 values based on growth rate were 13.73 and 27.54 mg/L, respectively. Based on the yield and compared to the negative controls: In all loading rates tested, the influence on algal growth ranged from 100.5 % inhibition to 23.2 % enhancement. Based on the average growth rates and compared to the negative controls: In all loading rates tested, the influence on algal growth ranged from 100.0 % inhibition to 3.9 % enhancement. No change in the colour of the media and no precipitation of test substance was noted during the incubation period. Microscopic evaluation of the inoculum at the start of the test and of the algae at the end of the test revealed normal appearance of the algae.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae toxicity study.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-03-09 until 2000-04-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA Guideline 712-C-96-164: OPPTS 850.5400 (1996, Public Draft) based on 40 CFR 797.1050 Algal Acute Toxicity Test, OPP 123-2

Deviations:

yes

Remarks:

, see in the field below

Qualifier:

according to guideline

Guideline:

other: Growth and Reproduction of Aquatic Plants, Tier 2, EPA report 540/0-82-020, 1982

Deviations:

yes

Remarks:

, see in the field below

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

, 1984

Deviations:

yes

Remarks:

, see in the field below

Principles of method if other than guideline:

In the control the cell density increased form 1 e04 cells/mL at the start of the test to 105 e04 cells/mL after 72 hours. To the end of the test (96 hours) the cell density further increased to 185 e04 cells/mL. The recommended cell density of 350 e04 cells/mL after an exposure period of 96 h was not reached at the light conditions given in the guideline. However, algal growth was high enough to determine the toxic effect of the test item Cyanamid L500 to Pseudokirchneriella subcapitata. According to OPP 123.2, primarily the cell density at the start of the test is important and therefore, the validity criteria are met.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Not indicated

Vehicle:

no

Details on test solutions:

Not indicated

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Not indicated

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observations

Hardness:

Not indicated

Test temperature:

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges.

pH:

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges.

Dissolved oxygen:

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges.

Salinity:

Not indicated

Nominal and measured concentrations:

0.16, 0.32, 0.63, 1.3, 2.5, 5.0, 10.0, and 20.0 mg of a 51.1 % (w/w) aqueous solution of cyanamide/L.
Mean measured concentrations of the 51.1 % (w/w) aqueous solution of cyanamide were 2.24, 4.26, 8.76, and 17.7 mg/L and ranged from 85 to 90 % of nominal concentrations.

Details on test conditions:

Algal cultures in synthetic test water, prepared according to the mentioned test guidelines. Six controls and three replicates at 0.16, 0.32, 0.63, 1.3, 2.5, 5.0, 10.0, and 20.0 mg of a 51.1 % (w/w) aqueous solution of Cyanamide/L were incubated for 96 hours and cell counts were taken at 24-hour intervals.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

16.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: There was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: There was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: There was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: There was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant.

Details on results:

The microscopic examination of the algal cells after 96 hours test period showed no difference between the algae growing at the test level of nominal 20 mg/L and the algal cells in the control. The shape and size of the algal cell growth up to this test concentration was obviously not affected.
Growth inhibition data obtained with the 51.1 % (w/w) aqueous solution of Cyanamide on Pseudokirchneriella subcapitata were as follows: For biomass, the 96-hour EbC50 was 13.2 mg of the 51.1 % (w/w) aqueous solution of Cyanamide/L (6.7 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of Cyanamide/L (2.6 mg as/L). Considering the growth rate the following data were obtained: The 96-hour ErC50 was 32.5 mg of the 51.1 % (w/w) aqueous solution of Cyanamide/L (16.6 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of Cyanamide/L (2.6 mg as/L).
However, there was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant (see in the table above "effect concentrations").

Results with reference substance (positive control):

No positive control

Reported statistics and error estimates:

Not indicated

Algal growth inhibition following exposure of Pseudokirchneriella subcapitata to the 51.1 % (w/w) aqueous solution of cyanamide for 96 hours:

Nominal concentrations of the 51.1% (w/w) aqueous solution of cyanamide (mg /L)	Mean cell density^1 (cells/mL)	% Inhibition^2
		Cell density	Growth rate	Area under the growth curve
Water control	1.84 x 10^6	---	---	---
0.16	2.12 x 10^6	-15.2	-2.7	-16.1
0.32	2.09 x 10^6	1.4	-2.4	-11.8
0.63	1.83 x 10^6	12.4	0.1	-1.3
1.3	1.91 x 10^6	-4.4	-0.6	-4.9
2.5	1.95 x 10^6	-2.1	-0.9	-9.6
5.0	1.91 x 10^6	2.1	-0.6	1.9
10.0	1.32 x 10^6	30.9	6.4*	36.2*
20.0	0.52 x 10^6	60.6	24.2*	75.1*

¹= test was started by inoculation of 10000 algal cells / mL

²= increase in cell density, growth rate and area under the growth curve relative to the control

* = significantly different from control according to the Dunnett test, p < 0.05.

Toxicity (96 h) of cyanamide toPseudokirchneriellasubcapitata– Summary of endpoints:

Endpoint	Nominal concentration of 51.1 % (w/w) aqueous solution of cyanamide
Cell density (96 h):
EbC50	13.2 mg/L (6.7 mg as/L)
No observed effect concentration (NOEC)	5.0 mg/L (2.6 mg as/L)
Growth rate:
ErC50	32.5 mg/L (16.6 mg as/L)
No observed effect concentration (NOEC)	5.0 mg/L (2.6 mg as/L)

 

Validity criteria fulfilled:

not specified

Conclusions:

Growth inhibition data obtained with the 51.1 % (w/w) aqueous solution of cyanamide on Pseudokirchneriella subcapitata were as follows: For biomass, the 96-hour EbC50 was 13.2 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (6.7 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (2.6 mg as/L). Considering the growth rate the following data were obtained: The 96-hour ErC50 was 32.5 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (16.6 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (2.6 mg as/L). However, there was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant (see in the table above "effect concentrations").

Executive summary:

The effect of SKW cyanamide L 500 (a 51.1 % (w/w) aqueous solution of cyanamide) on Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) was determined using algal cultures in synthetic test water, prepared according to the mentioned test guidelines. Six controls and three replicates at 0.16, 0.32, 0.63, 1.3, 2.5, 5.0, 10.0, and 20.0 mg of a 51.1 % (w/w) aqueous solution of cyanamide/L were incubated for 96 hours and cell counts were taken at 24-hour intervals. The samples of the test medium were analysed by a HPLC method using an UV/VIS detection. All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges. Mean measured concentrations of the 51.1 % (w/w) aqueous solution of cyanamide were 2.24, 4.26, 8.76, and 17.7 mg/L and ranged from 85 to 90 % of nominal concentrations. In the test medium, the test item was sufficiently stable during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test item. The microscopic examination of the algal cells after 96 hours test period showed no difference between the algae growing at the test level of nominal 20 mg/L and the algal cells in the control. The shape and size of the algal cell growth up to this test concentration was obviously not affected.However, there was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant. The 72-h effect values were calculated by the RMS and are as follows:EbC50:12.6 mg/L (6.5 mg as/L),corresponding NOEC:5.0 mg/L (2.6 mg as/L),ErC50: 28.8 mg/L (14.7 mg as/L), corresponding NOEC:5.0 mg/L (2.6 mg as/L).

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

, adopted April 1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Mean measured concentrations of the 49% (w/w) aqueous solution of cyanamide were 0.39, 0.72, 2.6, 4.85, 9.75, and 38.0 mg /L and ranged from 58 to 113 % of nominal concentrations.

Vehicle:

no

Details on test solutions:

Not indicated

Test organisms (species):

Scenedesmus capricornutum

Details on test organisms:

Not indicated

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

90.5 h

Post exposure observation period:

No post exposure observations

Hardness:

Not indicated

Test temperature:

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges.

pH:

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges.

Dissolved oxygen:

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges.

Salinity:

Freshwater

Nominal and measured concentrations:

1.2, 2.2, 7.0, 12.3, 22.0, and 70.4 mg of a 49% (w/w) aqueous solution of cyanamide/L (equivalent to 0.6, 1.08, 3.45, 6.0, 10.8, and 34.5 mg of the pure ai/L).
Mean measured concentrations of the 49% (w/w) aqueous solution of cyanamide were 0.39, 0.72, 2.6, 4.85, 9.75, and 38.0 mg /L.

Details on test conditions:

The effect of a 49% (w/w) aqueous solution of cyanamide on Selenastrum capricornutum was determined using algal cultures with a nutrient medium, described in the Netherlands Standard NPR 6505. Six controls and three replicates at 1.2, 2.2, 7.0, 12.3, 22.0, and 70.4 mg of a 49% (w/w) aqueous solution of cyanamide/L (equivalent to 0.6, 1.08, 3.45, 6.0, 10.8, and 34.5 mg of the pure ai/L), were incubated for 90.5 hours and cell counts were taken at 20.5, 43.5, 67.0, and 90.5 hours.

Reference substance (positive control):

no

Duration:

90.5 h

Dose descriptor:

EC50

Effect conc.:

13.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

90.5 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Results with reference substance (positive control):

No positive control

Reported statistics and error estimates:

Not indicated

In the test medium, the test item was sufficiently stable during the test period of 90.5 hours at concentrations higher than 6.0 mg aqueous solution of cyanamide/L. Since the EC₅₀-values are determined to be higher than 6.0 mg/L, the test results are based on nominal test concentrations.

Microscopic inspection of the morphology of algal cells in the cultures revealed that some cells showed an abnormal morphology at the test concentration of 34.5 mg/L. The viability of the cultures at the end of the test was assessed by staining with Evans-blue. It appeared that at all concentrations almost all cells were viable (some dead cells were found only at 3.45 mg/L).

Summary of algal growth inhibition following exposure of Selenastrum capricornutumto the 49% (w/w) aqueous solution of cyanamide for 90.5 hours:

Nominal concentrations of a 49% (w/w) aqueous solution of cyanamide (mg/L)	Mean cell density^*(cells/mL)	% Inhibition of cell density
Water control	4.4 x 10^6	---
0	4.2 x 10^6	4.6
1.2	4.3 x 10^6	-2.4
2.2	4.3 x 10^6	0
7.0	3.6 x 10^6	16.3
12.3	2.3 x 10^6	36.1
22.0	0.6 x 10^6	73.9
70.4	0.05 x 10^6	91.7

---    = indicates that the cell density is not inhibited

*       = test was started by inoculation of 10000 algal cells / ml

Toxicity to algae–Summary of endpoints:

Test substance	49 % (w/w) aqueous solution of cyanamide
Test object	Selenastrum capricornutum
Test duration	90.5 h
EC50	27.5 mg/L
	13.5 mg of the pure ai/L
No observed effect concentration (NOEC)	2.2 mg/L
	1.0 mg of the pure ai/L

Validity criteria fulfilled:

not specified

Conclusions:

Growth inhibition data obtained with aqueous hydrogen cyanamide on Selenastrum capricornutum were as follows:
Growth rate: 90.5-hour EC50 = 27.5 mg of the 49% (w/w) aqueous solution of cyanamide = 13.5 mg of the pure ai/L
90.5-hour NOEC = 2.2 mg of the 49% (w/w) aqueous solution of cyanamide = 1.0 mg of the pure ai/L

Executive summary:

The effect of a 49% (w/w) aqueous solution of cyanamide on Selenastrum capricornutum was determined using algal cultures with a nutrient medium, described in the Netherlands Standard NPR 6505. Six controls and three replicates at 1.2, 2.2, 7.0, 12.3, 22.0, and 70.4 mg of a 49% (w/w) aqueous solution of cyanamide/L (equivalent to 0.6, 1.08, 3.45, 6.0, 10.8, and 34.5 mg of the pure ai/L), were incubated for 90.5 hours and cell counts were taken at 20.5, 43.5, 67.0, and 90.5 hours.

Mean measured concentrations of the 49% (w/w) aqueous solution of cyanamide were 0.39, 0.72, 2.6, 4.85, 9.75, and 38.0 mg /L and ranged from 58 to 113 % of nominal concentrations.

In the test medium, the test item was sufficiently stable during the test period of 90.5 hours at concentrations higher than 6.0 mg aqueous solution of cyanamide/L. Since the EC50-values are determined to be higher than 6.0 mg/L, the test results are based on nominal test concentrations.

Microscopic inspection of the morphology of algal cells in the cultures revealed that some cells showed an abnormal morphology at the test concentration of 34.5 mg/L. The viability of the cultures at the end of the test was assessed by staining with Evans-blue. It appeared that at all concentrations almost all cells were viable (some dead cells were found only at 3.45 mg/L).

Growth inhibition data obtained with aqueous hydrogen cyanamide on Selenastrum capricornutum were as follows:

Growth rate: 90.5-hour EC50 = 27.5 mg of the 49% (w/w) aqueous solution of cyanamide/L = 13.5 mg of the pure ai/L

90.5-hour NOEC = 2.2 mg of the 49% (w/w) aqueous solution of cyanamide/L = 1.0 mg of the pure ai/L.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

2000-03-13 until 2000-08-31

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This growth inhibition test with Anabaena flos-aquae is not valid because of the following reason: Growth rate is the preferred evaluation parameter from an algae growth inhibition test. To enable a proper growth rate evaluation the control culture must show exponential growth over the whole exposure period. However, the control replicates of this test do not grow exponentially: According to OECD guideline 201 the specific growth rate for Anabaena flos-aquae should be between 1.1 – 1.4 per day. In this test the daily growth rates are 2.6 between day 0 and 1, 0.33 between day 1-2 and 0.44 between day 2-3. The mean coefficient of variation for section-by-section specific growth rate is 114%. Consequently this test cannot be used for the effect assessment for cyanamide and a reliability of 3 is appropriate to this study.This study was given a reliability factor of 3 in the frame of the approval of cyanamide as an existing biocidal active substance. As there is another valid algal growth inhibition test available (Pseudokirchneriella subcapitata) for cyanamide, a repeat of the study was not required.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

, (1984)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

, (1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Not indicated

Vehicle:

no

Details on test solutions:

not indicated

Test organisms (species):

Anabaena flos-aquae

Details on test organisms:

Not indicated

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period

Hardness:

Not indicated

Test temperature:

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges.

pH:

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges.

Dissolved oxygen:

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges.

Salinity:

Not indicated

Nominal and measured concentrations:

0.1, 0.21, 0.46, 1.0, 2.1 and 4.6 mg of the 50.6 % (w/w) aqueous solution of cyanamide/L.
Mean measured concentrations of the 50.6 % (w/w) aqueous solution of cyanamide were 0.09, 0.18, 0.39, 0.95, 2.02 and 4.35 mg/L and ranged from 79 to 104 % of nominal concentrations

Details on test conditions:

The effect of a 50.6 % (w/w) aqueous solution of cyanamide on Anabaena flos-aquae was determined using algal cultures in nutrient medium.
Three replicates were done for each concentration. Anabaena flos-aquae was incubated with the test item for 72 hours and cell counts were taken at 24-hour intervals.
The test item in the test medium was sufficiently stable during the test period of 72 hours.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.67 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.65 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Growth inhibition data obtained with a 50.6 % (w/w) aqueous solution of cyanamide (mg/L) on Anabaena flos-aquae were as follows: For biomass, the 72-hour EbC50 was 1.33 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.67 mg as/L) with a corresponding 72-hour NOEC of 0.1 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.05 mg as/L). With respect to growth rate, the 72-hour ErC50 was 1.29 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.65 mg as/L) and the corresponding 72-hour NOEC was 0.21 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.11 mg as/L).

Results with reference substance (positive control):

No positive control

Reported statistics and error estimates:

Test results are significantly different from control according to the Dunnett-test criteria, p < 0.05

Summary of algal growth inhibition following exposure of Anabaena flos-aquae to a 50.6% (w/w) aqueous solution of cyanamide for 72 hours:

Nominal concentrations of the 50.6% (w/w) aqueous solution of cyanamide (mg/L)	Mean cell density (cells/mL)^1	% Inhibition
		Cell density	Growth rate	Area under the growth curve
Water control	0.29 x 10^6	---	---	---
0.1	0.28 x 10^6	3.4	0.6	3.5
0.21	0.19 x 10^6	34.4	11.4	17.7*
0.46	0.13 x 10^6	55.2	24.4*	29.5*
1.0	0.07 x 10^6	75.9	41.1*	42.8*
2.1	0.04 x 10^6	86.2	63.2*	60.8*
4.6	0.007 x 10^6	97.6	119.9*	74.8*

  ¹Cell density at test start: 10000 algal cells/mL

* Significantly different from control according to the Dunnett-test criteria, p < 0.05, test was conducted for the growth rate and the area under the growth curve.

Toxicity (72 h) of Cyanamide toAnabaena flos-aquae– Summary of endpoints:

Endpoint	Nominal concentration of a 50.6 % (w/w) aqueous solution of cyanamide
Biomass:
EbC50	1.33 mg/L (0.67 mg as/L)
No observed effect concentration (NOEC)	0.1 mg/L (0.05 mg as/L)
Growth rate:
ErC50	1.29 mg/L (0.65 mg as/L)
No observed effect concentration (NOEC)	0.21 mg/L (0.11 mg as/L)

 

Validity criteria fulfilled:

no

Conclusions:

Growth inhibition data obtained with a 50.6 % (w/w) aqueous solution of cyanamide (mg/L) on Anabaena flos-aquae were as follows: For biomass, the 72-hour EbC50 was 1.33 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.67 mg as/L) with a corresponding 72-hour NOEC of 0.1 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.05 mg as/L). With respect to growth rate, the 72-hour ErC50 was 1.29 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.65 mg as/L) and the corresponding 72-hour NOEC was 0.21 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.11 mg as/L).

Executive summary:

The effect of a 50.6 % (w/w) aqueous solution of cyanamide on Anabaena flos-aquae was determined using algal cultures in nutrient medium. Three replicates at 0.1, 0.21, 0.46, 1.0, 2.1 and 4.6 mg of the 50.6 % (w/w) aqueous solution of cyanamide/L, were incubated for 72 hours and cell counts were taken at 24-hour intervals. The samples of the test medium were analysed by an HPLC method using an UV-VIS detector. All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges. Mean measured concentrations of the 50.6 % (w/w) aqueous solution of cyanamide were 0.09, 0.18, 0.39, 0.95, 2.02 and 4.35 mg/L and ranged from 79 to 104 % of nominal concentrations. The test item in the test medium was sufficiently stable during the test period of 72 hours. Growth inhibition data obtained with a 50.6 % (w/w) aqueous solution of cyanamide (mg/L) on Anabaena flos-aquae were as follows: For biomass, the 72-hour EbC50 was 1.33 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.67 mg as/L) with a corresponding 72-hour NOEC of 0.1 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.05 mg as/L). With respect to growth rate, the 72-hour ErC50 was 1.29 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.65 mg as/L) and the corresponding 72-hour NOEC was 0.21 mg of a 50.6 % (w/w) aqueous solution of cyanamide/L (0.11 mg as/L).

The study was not regarded relevant for classification and labeling as this test has significant deficiencies and an exotic blue-green algae species was tested. Two studies with green algae are considered as reliable results for derivation of classification and labeling.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Upon dissolution in water calcium cyanamide is fast transformed to hydrogen cyanamide.
Therefore, ecotoxicity in natural aquatic environments can be expressed in terms of cyanamide, irrespective of the substance constituting the exposure source. This is supported by experimental results: After stoichiometric correction of test concentrations, toxic effect values of cyanamide and calcium cyanamide are very similar in all standard test organisms. Thus, read-across from cyanamide to calcium cyanamide is justified for aquatic environmental endpoints.
For agricultural applications, calcium cyanamide is formulated as granules (PERLKA) that only slowly dissolve in a soil environment. Exposure of the aquatic environment will be exclusively due to runoff from agricultural fields. As above, cyanamide is the chemical moiety relevant for exposure due to rapid transformation from calcium cyanamide to cyanamide.
(Please note: direct release of the product to surface waters is strictly advised against, thus not a relevant exposure pathway.)
For detailled description where read across is used/recommended and where it is preferrable to refain from read across, please see section 13.2 "read across justification for environmental endpoints" and "Scientific rationale for not using cyanamide as read-across substance for calcium cyanamide on toxicological endpoints"

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

I

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

16.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: There was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: There was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: There was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: There was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant.

Details on results:

The microscopic examination of the algal cells after 96 hours test period showed no difference between the algae growing at the test level of nominal 20 mg/L and the algal cells in the control. The shape and size of the algal cell growth up to this test concentration was obviously not affected.
Growth inhibition data obtained with the 51.1 % (w/w) aqueous solution of Cyanamide on Pseudokirchneriella subcapitata were as follows: For biomass, the 96-hour EbC50 was 13.2 mg of the 51.1 % (w/w) aqueous solution of Cyanamide/L (6.7 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of Cyanamide/L (2.6 mg as/L). Considering the growth rate the following data were obtained: The 96-hour ErC50 was 32.5 mg of the 51.1 % (w/w) aqueous solution of Cyanamide/L (16.6 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of Cyanamide/L (2.6 mg as/L).
However, there was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant (see in the table above "effect concentrations").

Results with reference substance (positive control):

No positive control

Reported statistics and error estimates:

Not indicated

Algal growth inhibition following exposure of Pseudokirchneriella subcapitata to the 51.1 % (w/w) aqueous solution of cyanamide for 96 hours:

Nominal concentrations of the 51.1% (w/w) aqueous solution of cyanamide (mg /L)	Mean cell density^1 (cells/mL)	% Inhibition^2
		Cell density	Growth rate	Area under the growth curve
Water control	1.84 x 10^6	---	---	---
0.16	2.12 x 10^6	-15.2	-2.7	-16.1
0.32	2.09 x 10^6	1.4	-2.4	-11.8
0.63	1.83 x 10^6	12.4	0.1	-1.3
1.3	1.91 x 10^6	-4.4	-0.6	-4.9
2.5	1.95 x 10^6	-2.1	-0.9	-9.6
5.0	1.91 x 10^6	2.1	-0.6	1.9
10.0	1.32 x 10^6	30.9	6.4*	36.2*
20.0	0.52 x 10^6	60.6	24.2*	75.1*

¹= test was started by inoculation of 10000 algal cells / mL

²= increase in cell density, growth rate and area under the growth curve relative to the control

* = significantly different from control according to the Dunnett test, p < 0.05.

Toxicity (96 h) of cyanamide toPseudokirchneriellasubcapitata– Summary of endpoints:

Endpoint	Nominal concentration of 51.1 % (w/w) aqueous solution of cyanamide
Cell density (96 h):
EbC50	13.2 mg/L (6.7 mg as/L)
No observed effect concentration (NOEC)	5.0 mg/L (2.6 mg as/L)
Growth rate:
ErC50	32.5 mg/L (16.6 mg as/L)
No observed effect concentration (NOEC)	5.0 mg/L (2.6 mg as/L)

 

Validity criteria fulfilled:

not specified

Conclusions:

Growth inhibition data obtained with the 51.1 % (w/w) aqueous solution of cyanamide on Pseudokirchneriella subcapitata were as follows: For biomass, the 96-hour EbC50 was 13.2 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (6.7 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (2.6 mg as/L). Considering the growth rate the following data were obtained: The 96-hour ErC50 was 32.5 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (16.6 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (2.6 mg as/L). However, there was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant (see in the table above "effect concentrations").

Upon dissolution in water calcium cyanamide is fast transformed to hydrogen cyanamide.
Therefore, ecotoxicity in natural aquatic environments can be expressed in terms of cyanamide, irrespective of the substance constituting the exposure source. This is supported by experimental results: After stoichiometric correction of test concentrations, toxic effect values of cyanamide and calcium cyanamide are very similar in all standard test organisms. Thus, read-across from cyanamide to calcium cyanamide is justified for aquatic environmental endpoints.
For agricultural applications, calcium cyanamide is formulated as granules (PERLKA) that only slowly dissolve in a soil environment. Exposure of the aquatic environment will be exclusively due to runoff from agricultural fields. As above, cyanamide is the chemical moiety relevant for exposure due to rapid transformation from calcium cyanamide to cyanamide.
(Please note: direct release of the product to surface waters is strictly advised against, thus not a relevant exposure pathway.)
For detailled description where read across is used/recommended and where it is preferrable to refain from read across, please see section 13.2 "read across justification for environmental endpoints" and "Scientific rationale for not using cyanamide as read-across substance for calcium cyanamide on toxicological endpoints"

Executive summary:

The effect of SKW cyanamide L 500 (a 51.1 % (w/w) aqueous solution of cyanamide) on Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) was determined using algal cultures in synthetic test water, prepared according to the mentioned test guidelines. Six controls and three replicates at 0.16, 0.32, 0.63, 1.3, 2.5, 5.0, 10.0, and 20.0 mg of a 51.1 % (w/w) aqueous solution of cyanamide/L were incubated for 96 hours and cell counts were taken at 24-hour intervals. The samples of the test medium were analysed by a HPLC method using an UV/VIS detection. All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges. Mean measured concentrations of the 51.1 % (w/w) aqueous solution of cyanamide were 2.24, 4.26, 8.76, and 17.7 mg/L and ranged from 85 to 90 % of nominal concentrations. In the test medium, the test item was sufficiently stable during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test item. The microscopic examination of the algal cells after 96 hours test period showed no difference between the algae growing at the test level of nominal 20 mg/L and the algal cells in the control. The shape and size of the algal cell growth up to this test concentration was obviously not affected.However, there was no exponential growth of the algae between 72 and 96 hours. Therefore, effect values related to the 72-hours growth period are regarded relevant. The 72-h effect values were calculated by the RMS and are as follows:EbC50:12.6 mg/L (6.5 mg as/L),corresponding NOEC:5.0 mg/L (2.6 mg as/L),ErC50: 28.8 mg/L (14.7 mg as/L), corresponding NOEC:5.0 mg/L (2.6 mg as/L).

This information is used in a read-across approach in the assessment of the target substance.

For detailled description where read across is used/recommended and where it is preferrable to refain from read across, please see section 13.2 "read across justification for environmental endpoints" and "Scientific rationale for not using cyanamide as read-across substance for calcium cyanamide on toxicological endpoints"

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Upon dissolution in water calcium cyanamide is fast transformed to hydrogen cyanamide.
Therefore, ecotoxicity in natural aquatic environments can be expressed in terms of cyanamide, irrespective of the substance constituting the exposure source. This is supported by experimental results: After stoichiometric correction of test concentrations, toxic effect values of cyanamide and calcium cyanamide are very similar in all standard test organisms. Thus, read-across from cyanamide to calcium cyanamide is justified for aquatic environmental endpoints.
For agricultural applications, calcium cyanamide is formulated as granules (PERLKA) that only slowly dissolve in a soil environment. Exposure of the aquatic environment will be exclusively due to runoff from agricultural fields. As above, cyanamide is the chemical moiety relevant for exposure due to rapid transformation from calcium cyanamide to cyanamide.
(Please note: direct release of the product to surface waters is strictly advised against, thus not a relevant exposure pathway.)
For detailled description where read across is used/recommended and where it is preferrable to refain from read across, please see section 13.2 "read across justification for environmental endpoints" and "Scientific rationale for not using cyanamide as read-across substance for calcium cyanamide on toxicological endpoints"

Reason / purpose for cross-reference:

read-across source

Duration:

90.5 h

Dose descriptor:

EC50

Effect conc.:

13.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

90.5 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Results with reference substance (positive control):

No positive control

Reported statistics and error estimates:

Not indicated

In the test medium, the test item was sufficiently stable during the test period of 90.5 hours at concentrations higher than 6.0 mg aqueous solution of cyanamide/L. Since the EC₅₀-values are determined to be higher than 6.0 mg/L, the test results are based on nominal test concentrations.

Microscopic inspection of the morphology of algal cells in the cultures revealed that some cells showed an abnormal morphology at the test concentration of 34.5 mg/L. The viability of the cultures at the end of the test was assessed by staining with Evans-blue. It appeared that at all concentrations almost all cells were viable (some dead cells were found only at 3.45 mg/L).

Summary of algal growth inhibition following exposure of Selenastrum capricornutumto the 49% (w/w) aqueous solution of cyanamide for 90.5 hours:

Nominal concentrations of a 49% (w/w) aqueous solution of cyanamide (mg/L)	Mean cell density^*(cells/mL)	% Inhibition of cell density
Water control	4.4 x 10^6	---
0	4.2 x 10^6	4.6
1.2	4.3 x 10^6	-2.4
2.2	4.3 x 10^6	0
7.0	3.6 x 10^6	16.3
12.3	2.3 x 10^6	36.1
22.0	0.6 x 10^6	73.9
70.4	0.05 x 10^6	91.7

---    = indicates that the cell density is not inhibited

*       = test was started by inoculation of 10000 algal cells / ml

Toxicity to algae–Summary of endpoints:

Test substance	49 % (w/w) aqueous solution of cyanamide
Test object	Selenastrum capricornutum
Test duration	90.5 h
EC50	27.5 mg/L
	13.5 mg of the pure ai/L
No observed effect concentration (NOEC)	2.2 mg/L
	1.0 mg of the pure ai/L

Validity criteria fulfilled:

not specified

Conclusions:

Growth inhibition data obtained with aqueous hydrogen cyanamide on Selenastrum capricornutum were as follows:
Growth rate: 90.5-hour EC50 = 27.5 mg of the 49% (w/w) aqueous solution of cyanamide = 13.5 mg of the pure ai/L
90.5-hour NOEC = 2.2 mg of the 49% (w/w) aqueous solution of cyanamide = 1.0 mg of the pure ai/L

Upon dissolution in water calcium cyanamide is fast transformed to hydrogen cyanamide.
Therefore, ecotoxicity in natural aquatic environments can be expressed in terms of cyanamide, irrespective of the substance constituting the exposure source. This is supported by experimental results: After stoichiometric correction of test concentrations, toxic effect values of cyanamide and calcium cyanamide are very similar in all standard test organisms. Thus, read-across from cyanamide to calcium cyanamide is justified for aquatic environmental endpoints.
For agricultural applications, calcium cyanamide is formulated as granules (PERLKA) that only slowly dissolve in a soil environment. Exposure of the aquatic environment will be exclusively due to runoff from agricultural fields. As above, cyanamide is the chemical moiety relevant for exposure due to rapid transformation from calcium cyanamide to cyanamide.
(Please note: direct release of the product to surface waters is strictly advised against, thus not a relevant exposure pathway.)
For detailled description where read across is used/recommended and where it is preferrable to refain from read across, please see section 13.2 "read across justification for environmental endpoints" and "Scientific rationale for not using cyanamide as read-across substance for calcium cyanamide on toxicological endpoints"

Executive summary:

The effect of a 49% (w/w) aqueous solution of cyanamide on Selenastrum capricornutum was determined using algal cultures with a nutrient medium, described in the Netherlands Standard NPR 6505. Six controls and three replicates at 1.2, 2.2, 7.0, 12.3, 22.0, and 70.4 mg of a 49% (w/w) aqueous solution of cyanamide/L (equivalent to 0.6, 1.08, 3.45, 6.0, 10.8, and 34.5 mg of the pure ai/L), were incubated for 90.5 hours and cell counts were taken at 20.5, 43.5, 67.0, and 90.5 hours.

Mean measured concentrations of the 49% (w/w) aqueous solution of cyanamide were 0.39, 0.72, 2.6, 4.85, 9.75, and 38.0 mg /L and ranged from 58 to 113 % of nominal concentrations.

In the test medium, the test item was sufficiently stable during the test period of 90.5 hours at concentrations higher than 6.0 mg aqueous solution of cyanamide/L. Since the EC50-values are determined to be higher than 6.0 mg/L, the test results are based on nominal test concentrations.

Microscopic inspection of the morphology of algal cells in the cultures revealed that some cells showed an abnormal morphology at the test concentration of 34.5 mg/L. The viability of the cultures at the end of the test was assessed by staining with Evans-blue. It appeared that at all concentrations almost all cells were viable (some dead cells were found only at 3.45 mg/L).

Growth inhibition data obtained with aqueous hydrogen cyanamide on Selenastrum capricornutum were as follows:

Growth rate: 90.5-hour EC50 = 27.5 mg of the 49% (w/w) aqueous solution of cyanamide/L = 13.5 mg of the pure ai/L

90.5-hour NOEC = 2.2 mg of the 49% (w/w) aqueous solution of cyanamide/L = 1.0 mg of the pure ai/L.

This information is used in a read-across approach in the assessment of the target substance.

For detailled description where read across is used/recommended and where it is preferrable to refain from read across, please see section 13.2 "read across justification for environmental endpoints" and "Scientific rationale for not using cyanamide as read-across substance for calcium cyanamide on toxicological endpoints"

Description of key information

The ErC50 (72 h) of calcium cyanamide (technical grade, Kalkstickstoff) in algae (Pseudokirchneriella subcapitata) was determined to be 27.54 mg/L based on nominal concentrations (growth rate). The NOErC was determined to be 13.73 mg/L based on growth rate.

The sample tested contains 69.3 % calcium cyanamide. A re-calculation of the effects values to cyanamide results an ErC50 of 10.0 mg/L and a NOErC of 4.99 mg/L.

In addition to testing data with calcium cyanamide, information on the read-across substance cyanamide is considered:
The (acute) toxicity of cyanamide (50.6 %-51.1 % (w/w) aqueous solution of cyanamide) to aquatic algae (Pseudokirchneriella subcapitata), cyanobacteria (Anabaena flos-aquae) and green algae ( Scenedesmus capricornutum) was examined in three available studies. Tests with cyanobacteria (Hertl, 2000) were not regarded as relevant for classification and labelling as this test has significant deficiencies and other tests with the more relevant green algae species confirm EC50 values of higher than 10 mg/L in several tests.

In the key study (Seyfried, 2000), growth inhibition data obtained with the 51.1 % (w/w) aqueous solution of cyanamide on Pseudokirchneriella subcapitata were as follows:

- For biomass, the 72-hour EbC50 was 12.6 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (6.5 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (2.6 mg as/L).

- Considering the growth rate the following data were obtained: The 72-hour ErC50 was 28.8 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (14.7 mg as/L) with a corresponding NOEC of 5.0 mg of the 51.1 % (w/w) aqueous solution of cyanamide/L (2.6 mg as/L).

The study of Schoot-Uiterkamp (1988) examined the effect of a 49 % (w/w) aqueous solution of cyanamide on Selenastrum capricornutum using algal cultures with a nutrient medium in a 90.5 h test. The 90.5-hour ErC50 = 27.5 mg of the 49 % (w/w) aqueous solution of cyanamide (13.5 mg of the pure ai/L) and the corresponding 90.5-hour NOEC = 2.2 mg of the 49% (w/w) aqueous solution of cyanamide (1.0 mg of the pure ai/L).

As the key data for calcium cyanamide and cyanamide were conducted with the same algae species, the most sensitive ErC50 and NOErC from each study is carried forward as key values in the chemical safety assessment (reported as mg cyanamide/L).

Key value for chemical safety assessment

EC50 for freshwater algae:

10 mg/L

EC10 or NOEC for freshwater algae:

2.6 mg/L

Additional information

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to calcium cyanamide, technical grade (Kalkstickstoff) at nominal concentrations of 0; 0.74; 1.65; 3.27; 6.64; 13.73; 25.88; 49.95; 104.40; 200.25 mg/L under static conditions in accordance with EC regulation 761/2009 Part C.3 and the OECD-Guideline 201. The test substance has impurities and is of limited solubility in aqueous media. Therefore the algae were exposed to separate preparations at different loading rates of water accommodated fractions (WAFs).

The NOEC and EC50 values based on growth rate were 13.73 and 27.54 mg/L, respectively. These values correspond to 9.5 mg/L and 19.1 mg/L for pure calcium cyanamide.

Based on the yield and compared to the negative controls: In all loading rates tested, the influence on algal growth ranged from 100.5 % inhibition to 23.2 % enhancement. Based on the average growth rates and compared to the negative controls: In all loading rates tested, the influence on algal growth ranged from 100.0 % inhibition to 3.9 % enhancement. No change in the colour of the media and no precipitation of test substance was noted during the incubation period. Microscopic evaluation of the inoculum at the start of the test and of the algae at the end of the test revealed normal appearance of the algae.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae toxicity study.

Upon dissolution in water calcium cyanamide is fast transformed to hydrogen cyanamide.

Therefore, ecotoxicity in natural aquatic environments can be expressed in terms of cyanamide, irrespective of the substance constituting the exposure source. This is supported by experimental results: After stoichiometric correction of test concentrations, toxic effect values of cyanamide and calcium cyanamide are very similar in all standard test organisms. Thus, read-across from cyanamide to calcium cyanamide is justified for aquatic environmental endpoints.

For agricultural applications, calcium cyanamide is formulated as granules (PERLKA) that only slowly degrade in a soil environment. Exposure of the aquatic environment will be exclusively due to runoff from agricultural fields. As above, cyanamide is the chemical moiety relevant for exposure due to rapid transformation from calcium cyanamide to cyanamide.

(Please note: direct release of the product to surface waters is strictly advised against, thus not a relevant exposure pathway.

For further information on read-across for ecotoxicological endpoints please refer to the document in IUCLID section 13.)

Therefore, available information for cyanamide is considered in the assessment of calcium cyanamide in a read-across approach:

The (acute) toxicity of cyanamide (50.6 %-51.1 % (w/w) aqueous solution of cyanamide) to aquatic algae was examined in three available studies which were conducted according/similar to OECD Guideline 201. Results for cyanobacteria (Hertl, 2000) were not regarded as relevant for classification and labelling as this test has significant deficiencies and other test with the more relevant green algae species show EC50 values of higher than 10 mg/L.

The key study of Seyfried (2000, Doc. No., 823-003) examined the effect of SKW Cyanamid L500 (a 51.1 % (w/w) aqueous solution of cyanamide) on Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum, green algae) using algal cultures in synthetic test water in a 96-h test. As there was no exponential growth of the algae between 72 and 96 hours, the effect values related to the 72-hours growth period are regarded relevant. The 72-h effect values were calculated as follows:

-       EbC50 (b=biomass): 12.6 mg/L (6.5 mg ai/L), corresponding NOEC: 5.0 mg/L (2.6 mg ai/L);

-       ErC50 (r-growth rate): 28.8 mg/L (14.7 mg ai/L), corresponding NOEC: 5.0 mg/L (2.6 mg ai/L).

The study of Schoot-Uiterkamp (1988, Doc. No., 823-001) examined the effect of a 49 % (w/w) aqueous solution of cyanamide on Selenastrum capricornutum (green algae) using algal cultures with a nutrient medium in a 90.5 h test. The 90.5-hour ErC50 = 27.5 mg of the 49 % (w/w) aqueous solution of cyanamide (13.5 mg of the pure ai/L) and the corresponding 90.5-hour NOEC = 2.2 mg of the 49% (w/w) aqueous solution of cyanamide (1.0 mg of the pure ai/L).

The study of Hertl (2000, Doc. No., 823-004) was not regarded valid and relevant for classification and labelling because of the following reason:

Growth rate is the preferred evaluation parameter from an algae growth inhibition test. To enable a proper growth rate evaluation the control culture must show exponential growth over the whole exposure period. However, the control replicates of this test do not grow exponentially: According to OECD guideline 201 the specific growth rate for Anabaena flos-aquae should be between 1.1 – 1.4 per day. In this test the daily growth rates are 2.6 between day 0 and 1, 0.33 between day 1-2 and 0.44 between day 2-3. The mean coefficient of variation for section-by-section specific growth rate is 114 %. Consequently, this test cannot be used for the effect assessment for cyanamide and a reliability of 3 is appropriate to this study. This study was given a reliability factor of 3 in the frame of the approval of cyanamide as an existing biocidal active substance. As there is another valid algal growth inhibition test available (Pseudokirchneriella subcapitata) for cyanamide, a repeat of the study was not required.