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:

2000-03-09 until 2000-04-12

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:

, 1984

Deviations:

yes

Remarks:

, see in the field below

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

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

Conductivity:

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

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

recalculated by RMS during substance evaluation according to EU BPR

Effect conc.:

12.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Corresponding to 6.5 mg active ingredient/L (nominal). 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

Remarks:

recalculated by RMS during substance evaluation according to EU BPR

Effect conc.:

28.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Corresponding to 14.7 mg active ingredient/L (nominal). 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

Remarks:

recalculated by RMS during substance evaluation according to EU BPR

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Corresponding to 2.6 mg active ingredient/L (nominal). 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

Remarks:

recalculated by RMS during substance evaluation according to EU BPR

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Corresponding to 2.6 mg active ingredient/L (nominal). 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:

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

Key result

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

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:
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 2

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

Key result

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

Key result

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.

Description of key information

In a study similar to OECD 201 with Pseudokirchneriella subcapitata the 72-hour ErC50 and NOEC (growth rate) of the substance was determined to be 28.8 mg/L (nominal, corresponding to 14.7 mg a.i/L) and 5.0 mg/L (nominal, corresponding to 2.6 mg ai/L), respectively. The 72-hour EC50 and NOEC based on biomass were 12.6 mg/L (6.5 mg a.i./L) and 5.0 mg/L (2.6 mg a.i./L), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

14.7 mg/L

EC10 or NOEC for freshwater algae:

2.6 mg/L

Additional information

The (acute) toxicity of cyanamide (50.6 %-51.1 % (w/w) aqueous solution of cyanamide) to aquatic green algae and cyanobacteria was examined in three available studies which were conducted according/similar to OECD TG 201.

 

Key information

In the key study of Seyfried (2000, Doc. No., 823 -003) the effect of SKW Cyanamid L500 (a 51.1 % (w/w) aqueous solution of cyanamide) on Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum, green algae) was assessed using algal cultures in synthetic test water in a 96 h test. The method described in OECD TG 201 (1984), OPPTS 850.5400 (1996, Public Draft) and US EPA report 540/0-82-020, (1982) was followed. Algae were exposed to 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. In the negative 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. 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, 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 study of Seyfried (2000) has been reviewed during substance evaluation according to EU BPR. The Rapporteur Member State recalculated the 72-hour effect values to be: EC50 = 12.6 mg/L (6.5 mg ai/L) and NOEC = 5.0 mg/L (2.6 mg ai/L) based on biomass and EC50 = 28.8 mg/L (14.7 mg ai/L) and NOEC = 5.0 mg/L (2.6 mg a.i./L) based on growth rate. The study was performed according to scientifically accepted methods and is considered acceptable for the hazard and risk assessment according to Regulation (EC) No 1907/2006 (REACH).

 

 

Supporting information

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. Algae were exposed to nominal test item concentrations of 1.2, 2.2, 7.0, 12.3, 22.0, and 70.4 mg (49% (w/w) aqueous solution of cyanamide/L), which corresponds to 0.6, 1.08, 3.45, 6.0, 10.8, and 34.5 mg of the pure a.i./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.

 

The 90.5-hour ErC50 was 27.5 mg of the 49 % (w/w) aqueous solution of cyanamide (13.5 mg of the pure a.i./L) and the corresponding 90.5-hour NOEC was 2.2 mg of the 49% (w/w) aqueous solution of cyanamide (1.0 mg of the pure a.i./L).

The study of Hertl (2000, Doc. No., 823-004) was not regarded valid and relevant for classification and labeling 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 under EU BPR.

 

Conclusion

Information on the growth inhibiting effect of cyanamide to green algae and cyanobacteria is available. In the most current and reliable study (Seyfried, 2000) the 72-hour EC50 and NOEC based on biomass was determined to be 12.6 mg/L (nominal, corresponding to 6.5 mg a.i./L) and 5.0 mg/L (nominal, corresponding to 2.6 mg a.i./L). Based on growth rate the 72-hour EC50 and NOEC was found to be 28.8 mg/L (nominal, corresponding to 14.7 mg a.i/L) and 5.0 mg/L (nominal, corresponding to 2.6 mg ai/L), respectively. In accordance with the criteria given in Regulation (EC) 1272/2008 (CLP, , as amended for the eighteenth time in Regulation (EU) 2022/692) growth rate is the preferred evaluation parameter from an algae growth inhibition test. Therefore the 72-hour ErC50 and NOEC (growth rate) of 28.8 mg/L (nominal, corresponding to 14.7 mg a.i/L) and 5.0 mg/L (nominal, corresponding to 2.6 mg a.i./L) are considered as key values for the Hazard and Risk Assessment.