Calcium cyanamide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-03-17 to 2010-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Regulation (EC) 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 2004-04-13

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for chemical analysis were taken at the start and at the end of the test and were sent to the Test Site (Alzchem, Trostberg) 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 (Alzchem, Trostberg) was on dry ice.

Vehicle:

no

Details on test solutions:

The stock solutions for preparation of the reconstituted water according to ISO 6341 were obtained by MicroBioTests Inc. (Batch No. ISOD231109). The water was aerated and the pH, the dissolved oxygen concentration and the water hardness were checked before using.

Preparation of the test media
The test substance is a substance with impurities and is of limited solubility in aqueous media. Therefore the daphnia were exposed to separate preparations of 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 medium (reconstituted water) and stirred for 60 minutes in the dark. Not dissolved particles were allowed to precipitate and the surplus was filtrated (0.45 μm, Minisart, Sartorius) to obtain a clear solution which was used for the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

Neonates of Daphnia magna (Crustacea, Cladocera), less than 24 hours old, were obtained by hatching ephippia, supplied in the microbiotest "Daphtoxkit F TM " (Daphnia magna; Fa. Micro BioTests Inc; Kleimoer 15, B-9030 Mariakerke (Gent); BELGIUM). The ephippia were hatched in the laboratory in a petri dish containing reconstituted water in a temperature controlled room at approx. 20-22 °C under continuous illumination of approximately 6000 Lux. According to MicroBioTests Inc. the largest hatching occurs between 72 and 80 hours and the Daphnia must be collected at the latest 90 hours after initiation of hatching. The neonates were collected for this test at approximately 88 hours after initiation of hatching. Approximately 2 hours before the start of the test the neonates were fed with a suspension of Spirulina powder (MicroBioTests Inc.), as also in traditional Daphnia cultures the neonates have the opportunity to take up some feed particles.
Batch No. of the ephippia in the test: DM161209.
The batch of ephippia is provided together with a specification sheet with batch number and date of expiry. Additionally for confirmation of the sensitivity of the test animals of the used batch the results of a reference test with potassium dichromate are provided. 24 h EC50 of potassium dichromate: 1.13 mg/L for the batch used. The acceptable range of the EC50 24 h according to the OECD guideline 202 is 0.6 mg/L to 2.1 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

220 mg CaCO3/L

Test temperature:

20-21°C

pH:

Due to the basic nature of the test substance the pH of the relevant test media ranged from 7.4 to 9.9. The pH of the control medium was between 8.2 and 7.3.

Dissolved oxygen:

at least 8.5 mg/L

Salinity:

not applicable

Nominal and measured concentrations:

Nominal: 1.32, 2.55, 4.80, 11.00, 20.80, 42.80 mg/L
Measured: 1.40, 1.79, 4.12, 12.10, 21.07, 41.93 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
Disposable multiwell test plates with 30 test wells with a transparent lid, provided with the Daphtoxkit F. Each plate has four 15 mL wells (replicates A, B, C, D) for each of the control and the 5 concentrations. Additionally, for each concentration the plates are provided with "rinsing wells" to prevent dilution of the test substance during the transfer of the Daphnia from the hatching petri dish to the test wells.
- Aeration: No extra aeration, but slight circulation of air is still possible when the lid of the test plate is closed.

The "rinsing wells" and the wells for the replicates of each concentration and control respectively were filled with 10 mL medium each. For the test substance concentrations and for the control at least 20 actively swimming neonates were transferred with a micropipette from the hatching petri dish to the "rinsing well". Then 5 neonates from the "rinsing wells" were transferred to each of the 4 wells of the appropriate concentration.

Animal observation
24 and 48 hours after the start of the exposure to the test substance the neonates were observed for their swimming behaviour and other signs of stress or disease (e.g. trapping of daphnia at the water surface or discolouration). Animals not able to swim within approximately 15 s after gentle agitation of the test container were evaluated as immobilised. Animals trapped at the surface were counted. Only the mobile daphnia trapped on the water surface were regarded as trapped but not the immobilised ones.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CI = 5.1-7.0 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Quality criteria
- There was no immobilisation and no other signs of stress or disease (e.g. trapping of daphnia at the water surface or discolouration) in the negative control group at the end of the test. (guideline: maximum of 10 %).
- The dissolved oxygen concentration was higher than 3 mg/L at each determination.

Results with reference substance (positive control):

24 h EC50 of potassium dichromate: 1.13 mg/L for the batch used. The acceptable range of the EC50 24 h according to the OECD guideline 202 is 0.6 mg/L to 2.1 mg/L.

Reported statistics and error estimates:

The EC0/EL0 and EC100/EL100 values were estimated from the immobilisation data. Ten % immobilisation is regarded by the OECD and EU guidelines as limit of natural mortality in the control group and are tolerated. Ten % or less immobilisation in the test substance group are therefore not considered as relevant for the determination of the EC0/EL0, if appropriate.
The NOEC/NOEL is defined as the test substance concentration/loading rate below the lowest concentration/loading rate that did result in a relevant effect in a specific experiment, i.e. the NOEC/NOEL is the tested concentration next below the LOEC/LOEL. The NOEC/NOEL was estimated from the number of immobilised and trapped daphnia at 48 h. As for the control group, 10 % or less immobilised and/or trapped daphnia on the surface in the test substance group is not considered as relevant for the determination of the NOEC/NOEL, if appropriate.

Validity criteria fulfilled:

yes

Conclusions:

In a static immobilisation test in daphnia, the EC50 (48 h, daphnia magna) was determined to be 6 mg/L calcium cyanamide (Kalkstickstoff). The NOAEC (48 h) was 1.8 mg/L.

Executive summary:

The 48 –hr-acute toxicity of calcium cyanamide (Kalkstickstoff) to daphnia magna was studied under static conditions. Calcium cyanamide is a substance with impurities and is of limited solubility in aqueous media. Therefore the daphnia were exposed to separate preparations of different loading rates of water accommodated fractions (WAFs). Nominal loading rates of 1.32, 2.55, 4.80, 11.00, 20.80, and 42.80 mg per litre were obtained by stirring the respective amount of test substance in reconstituted water according to ISO 6341 for 60 minutes in the dark. Afterwards the not dissolved particles were removed by filtration (0.45 μm) and the clear filtrate was used for the test. Reconstituted water only was used for the control group. 20 neonates of Daphnia magna, hatched from ephippia and less than 24 hours old were exposed in each of the 6 test substance groups and in the control group. Exposure was 48 h in a static test.

Mortality/immobilization and sublethal effects were observed daily. The 48 – hour EC50 was 6.0 mg/L. The 48 – hr NOEC based on immobilization was 1.8 mg /L.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Results Synopsis

Test Organism Age: neonate daphnia

Test Type: Static

EC50: 6.0 mg/L, 95% C.I.: 5.1 to 7.0 mg/L

NOEL: 1.8 mg /L

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

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

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

4.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

The test concentrations were not measured during the test. A summary of the findings is presented in Table A7.4.1.2/01-1. There were no test concentration related immobility or mortal effects below 5.6 mg of a 49 % (w/w) aqueous solution of cyanamide/L. The highest concentration causing no immobility or mortality was 3.2 mg/L and the lowest concentration causing 100 % immobility or mortality was 18.0 mg/L.

Results with reference substance (positive control):

No reference substance

Reported statistics and error estimates:

Not indicated

Immobilisation of unfed Daphnia magna exposed to a 49 % (w/w) aqueous solution of cyanamide for 48 hours in an unaerated, static acute test:

Nominal concentration of a 49 % (w/w) aqueous solution of cyanamide (mg/L)	Mortality (No. dead/No. at test start)		Immobility (No. immobile/No. at test start
	24 hours	48 hours	24 hours	48 hours
Water Control	0/20	0/20	0/20	0/20
1.8	0/20	0/20	0/20	0/20
3.2	0/20	0/20	0/20	0/20
5.6	1/20	3/20	1/20	6/20
10.0	4/20	11/20	7/20	18/20
18.0	13/20	20/20	15/20	20/20
32.0	11/20	20/20	11/20	20/20
56.0	12/20	20/20	12/20	20/20
100.0	12/20	20/20	13/20	20/20
180.0	19/20	20/20	20/20	20/20
320.0	20/20	20/20	20/20	20/20

Acute toxicity (48 h) of Cyanamide to daphnids – Summary of endpoints:

Endpoint	Nominal concentration of 49 % (w/w) aqueous solution of cyanamide
EC50	6.5 mg/L (3.2 mg as/L)
LC50	8.8 mg/L (4.3 mg as/L)
No observed effect concentration (NOEC)	3.2 mg/L (1.6 mg as/L)

 

Validity criteria fulfilled:

not specified

Conclusions:

The 48-hour EC50 in Daphnia magna was 6.5 mg of a 49 % (w/w) aqueous solution of cyanamide/L (equivalent to 3.2 mg as/L), based on immobilisation of the test animals. The 48-hour LC50 were 8.8 mg/L, based on mortality. The NOEC is estimated to be 3.2 mg/L (equivalent to 1.6 mg as/L). Since it is difficult to differentiate between immobile and dead daphnids, immobility is used as the relevant endpoint.

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 acute toxicity of Alzodef (a 49 % (w/w) aqueous solution of cyanamide) to unfed Daphnia magna (< 24 hours old) was determined in an unaerated, static, 48-hour test. Treatments consisted of a diluted water control, and nominal concentrations of 1.8, 3.2, 5.6, 10.0, 18.0, 32.0, 56.0, 100.0, 180.0 and 320.0 mg of a 49 % (w/w) aqueous solution of cyanamide/L. Five daphnids were used per replicate. The test was performed with four replicates per treatment.

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges. There were no test concentration related immobility or mortal effects below 5.6 mg of a 49 % (w/w) aqueous solution of cyanamide/L. The highest concentration causing no immobility or mortality was 3.2 mg/L and the lowest concentration causing 100 % immobility or mortality was 18.0 mg/L.

The 48-hour EC50 in Daphnia magna was 6.5 mg of a 49 % (w/w) aqueous solution of cyanamide/L (equivalent to 3.2 mg as/L), based on immobilisation of the test animals. The 48-hour LC50 were 8.8 mg/L, based on mortality. The NOEC is estimated to be 3.2 mg/L (equivalent to 1.6 mg as/L). Since it is difficult to differentiate between immobile and dead daphnids, immobility is used as the relevant endpoint.

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

In a static immobilisation test (Seibersdorf, 2010), the EC50 (48 h, Daphnia magna) was determined to be 6 mg/L calcium cyanamide (Kalkstickstoff). The NOAEC (48 h) was 1.8 mg/L. The concentration of calcium cyanamide in the sample tested is 69.3 %.

The re-calculation to cyanamide, the transformation product triggering toxic effects, result in an EC50 of 2.2 mg/L and a NOAEC of 0.66 mg/L.

The acute toxicity of the read-across substance cyanamide (Alzodef: 49 % (w/w) aqueous solution of cyanamide) to Daphnia magna was examined in one available study (Adema, 1983). In this static, 48 hours test the calculated EC50 was 3.2 mg of pure active ingredient (ai) cyanamide/L, NOEC of 1.6 mg ai/L and a LC50 of 4.3 mg ai/L.

The most sensitive value of EC50 = 2.2 mg cyanamide/L is carried forward in the risk assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.2 mg/L

Additional 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 degrades 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"

The 48-hr-acute toxicity of calcium cyanamide, technical grade (Kalkstickstoff) to Daphnia magna was studied under static conditions. Calcium cyanamide is a substance with impurities and is of limited solubility in aqueous media. Therefore, the daphnia were exposed to separate preparations of different loading rates of water accommodated fractions (WAFs). Nominal loading rates of 1.32, 2.55, 4.80, 11.00, 20.80, and 42.80 mg per litre were obtained by stirring the respective amount of test substance in reconstituted water according to ISO 6341 for 60 minutes in the dark. Afterwards the not dissolved particles were removed by filtration (0.45 μm) and the clear filtrate was used for the test. Reconstituted water only was used for the control group. 20 neonates of Daphnia magna, hatched from ephippia and less than 24 hours old were exposed in each of the 6 test substance groups and in the control group. Exposure was 48 h in a static test.

Mortality/immobilization and sublethal effects were observed daily. The 48-hour EC50 was 6.0 mg/L. The 48-hr NOEC based on immobilization was 1.8 mg /L.

The concentration of calcium cyanamide in the sample tested is 69.3 %.

The re-calculation to cyanamide, the transformation product triggering toxic effects, result in an EC50 of 2.2 mg/L and a NOAEC of 0.66 mg/L.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

The acute toxicity of Alzodef (a 49 % (w/w) aqueous solution of cyanamide) to unfed Daphnia magna (< 24 hours old) was determined in an unaerated, static, 48-hour test. Treatments consisted of a diluted water control, and nominal concentrations of 1.8, 3.2, 5.6, 10.0, 18.0, 32.0, 56.0, 100.0, 180.0 and 320.0 mg of a 49 % (w/w) aqueous solution of cyanamide/L. Five daphnids were used per replicate. The test was performed with four replicates per treatment.

All chemical and physical parameters (dissolved oxygen concentration, pH, temperature) in the definitive test were within expected ranges. There were no test concentration related immobility or mortal effects below 5.6 mg of a 49 % (w/w) aqueous solution of cyanamide/L. The highest concentration causing no immobility or mortality was 3.2 mg/L and the lowest concentration causing 100 % immobility or mortality was 18.0 mg/L.

The 48-hour EC50 in Daphnia magna was 6.5 mg of a 49 % (w/w) aqueous solution of cyanamide/L (equivalent to 3.2 mg as/L), based on immobilisation of the test animals. The 48-hour LC50 were 8.8 mg/L, based on mortality. The NOEC is estimated to be 3.2 mg/L (equivalent to 1.6 mg as/L). Since it is difficult to differentiate between immobile and dead daphnids, immobility is used as the relevant endpoint.