Calcium cyanamide

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: BBA (Biologische Bundesanstalt für Land- und Forstwirtschaft, Braunschweig): Proposal for an OECD-Guideline (ECO 85, UPEC 15): test guideline for the assessment of toxicity to earthworms (Eisenia foetida Sav.), Laboratory test, Draft-BBA AP 3000 b u. 2600

Deviations:

not specified

GLP compliance:

not specified

Analytical monitoring:

not specified

Details on sampling:

not indicated

Vehicle:

no

Details on preparation and application of test substrate:

The test substance was added in 200 mL of a emulsion of dispersion of deionized water while stirring a quantity of moist artificial soil which was equal to 1 kg dry weight: 1.55 kg of damp substrate (1.0 kg dry weight) was added to each container. Each test batch was mixed separately in a household mixer.

Test organisms (species):

other: Eisenia foetida and Lumbricus terrestris

Animal group:

annelids

Details on test organisms:

Eisenia foetida:
The earthworm species used was E. foetida andrei. This species has been bred in the test laboratory for several years under the conditions described in the BBA (1981) proposal for an OECD guideline. The earthworms used in the tests were adult, and were more than 2 months old. Their average weight was between 300-400 mg.
Lumbricus terrestris:
The earthworms used were purchased from a local bait dealer and originated from golf courses in Canada. Sexually matured worms with 3000-42000 mg each were used.

Study type:

laboratory study

Substrate type:

natural soil

Total exposure duration:

14 d

Post exposure observation period:

no data

Test temperature:

Eisenia foetida: 22 +/- 1°C
Lumbricus terrestris: 10-15 °C

pH:

Eisenia foetida: 7 +/- 0.5
Lumbricus terrestris: no data

Moisture:

Eisenia foetida: 35 % of the dry weight of the substrate, equal to 25.9 % of the wet weight of the substrate
Lumbricus terrestris: the water holding capacity was 35 %.

Details on test conditions:

Eisenia foetida:
Treated substrate was poured into 1.5-2 L glass containers and 10 earthworms, which had been washed in water, were placed on top of the substrate. The containers were covered with fine mesh gauze and stored in an incubation chamber which was held at 22 +/- 1°C, had a relative humidity od 70-90 % and had a light-dark-cycle of 12:12 h.
After 7 days, 5 g of finely ground dried cattle manure were mixed into each test container and the water lost by evaporation was replaced. After 14 days, the number of living earthworms were determined by counting. Earthworms were considered dead if they did not respond to a gentle mechanical stimulus. The LC50 was calculated by probit analysis.

Lumbricus terrestris:
As food for the earthworms, maize flour was spread on top of the soil. To each test container 3 kg wet soil and 6 earthworms were added.

Nominal and measured concentrations:

Eisenia foetida: The concentrations 1000, 100, 10, 1 and 0.1 mg/kg of artificial soil (dry weight) were tested.
Lumbricus terrestris: no data

Reference substance (positive control):

yes

Remarks:

Other pesticides with positive responses were tested in parallel

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

115 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: Eisenia foetida

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

124 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: Lumbricus terrestris

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

ca. 182.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

calculated from 124 mg a.i./kg with the assumption of 68% calcium cyanamide in Kalkstickstoff (see statement in section 13)

Basis for effect:

mortality

Remarks on result:

other: Lumbricus terrestris

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

ca. 169.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

calculated from 115 mg a.i./kg with the assumption of 68% calcium cyanamide in Kalkstickstoff (see statement in section 13)

Basis for effect:

mortality

Remarks on result:

other: Eisenia foetida

Details on results:

no further details given

Results with reference substance (positive control):

no positive control

Reported statistics and error estimates:

no data

Validity criteria fulfilled:

not specified

Conclusions:

The acute earthworm LC50 of calcium cyanamide was determined to be 115 mg/kg dry soil for Eisenia foetida and 124 mg/kg dry soil for Lumbricus terrestris (results based on 100 % active ingredient).

Executive summary:

Acute toxicity of calcium cyanamide to earthworms, Eisenia foetida and Lumbricus terrestris, was determined in a 14-day soil exposure laboratory study conducted similar to OECD Guideline 207.

Three replicates of 6 or 10 clitellated adult earthworms were each exposed to doses in a geometric series, in order to determine a LC50 value. No toxic reference standard was tested. Worms were assessed for mortality and sublethal effects after 7 and 14 days of exposure.

Mortality results and sublethal effects were not reported for each concentration. The acute earthworm LC50 of calcium cyanamide was determined to be 115 mg/kg dry soil for Eisenia foetida and 124 mg/kg dry soil corresponding for Lumbricus terrestris.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: BBA guideline VI, 2-3 (1994)

Deviations:

not applicable

GLP compliance:

no

Specific details on test material used for the study:

Mean concentration of calcium cyanamide in PERLKA is 44 %.

Analytical monitoring:

no

Details on sampling:

No analyrical monitoring

Vehicle:

no

Details on preparation and application of test substrate:

PERLKA was applied by hand at 400 kg/ha. The plots were divided in longitudinal and latitudinal strips and the appropriate amount of PERLKA was applied in two rounds (lengthwise and crosswise).

Test organisms (species):

other: Allolobophora caliginosa, A. rosea, A. longa, A. chlorotica, Lumbricus terrestris, L. castaneus, L. rubellus, Octolasium lacteum

Animal group:

annelids

Details on test organisms:

Naturally occurring earthworm species, adults and juveniles.

Study type:

field study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

52 wk

Remarks:

Single application of PERLKA in April 1991. Earthworm sampling in May 1991 and June 1992.

Post exposure observation period:

Single application of PERLKA in April 1991. Earthworm sampling in May 1991 and June 1992.

Test temperature:

soil temperature 0.9 - 19.1°C in 1991 and 1.1 - 20°C in 1992

pH:

5 - 6

Moisture:

soil moisture at application: about 37 %
soil humidity at first sampling: 23.9 - 33.4 %, soil humidity at last sampling: 21.5 - 34.1 %

Details on test conditions:

The test field was grassland (old ray grass-white clover meadow) wiht 5 cuttings/year. The soil is a loamy brown earth rich in roots.
The trial plos were situated in the midst of a large greenland plot.
The trial area was 80 x 80 m with four plos (each 20 x 20 m) for the control and PERLKA. Samples were taken from the 14 x 14 m core areas of the plots in order to exclude any influence from neighbour plots.

Sampling was performed in May 1991 and in June 1992. In May 1991, 10 samples per plot were taken with the formalin method. In June 1992, 4 samples per plot were taken using a combined methodology (formalin method, hand picking of remaining worms and expulsion of earthworms from strata below 20 cm by formalin)

Measured parameters were earthworm numbers per species, total numbers of earthworms, total masses of earthworms and average earthworm weights per species

Nominal and measured concentrations:

nominal: 400 kg/ha (533.3 mg/kg soil)

Reference substance (positive control):

no

Key result

Duration:

52 wk

Dose descriptor:

NOEC

Effect conc.:

>= 213.2 - <= 239.85 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: abundance and biomass

Remarks on result:

other: The sample tested contains 40-45 % calcium cyanamide. The NOECof 533 mg/kg soil dw obtained for the test material was re-calculated to 100 % calcium cyanamide.

Key result

Duration:

52 wk

Dose descriptor:

NOEC

Effect conc.:

533 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: abundance and biomass

Duration:

52 wk

Dose descriptor:

other: NOER

Effect conc.:

400 other: kg/ha

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: abundance and biomass

Details on results:

No negative effects following a single application of 400 kg PERLKA/ha (533 mg PERLKA/kg soil) on earthworm abundance and biomass of earthworms were observed.

Reported statistics and error estimates:

There were no statistically significant effects in the treated groups when compared to the control (H-Test of Kruskal & Wallis, Tuckey & Kramer-test and Nemenyi-Test, 5% level)

Validity criteria fulfilled:

not applicable

Conclusions:

The application of 400 kg PERLKA/ha (533 mg PERLKA/kg soil, equal to 235 mg/kg pure calcium cyanamide) under natural conditions had no effect on earthworm populations one and 12 months after application.

Executive summary:

In a field study on grassland (meadow) PERLKA was tested for possible side effects on earthworms. A single broadcast application at a rate of 400 kg PERLKA/ha (533 mg PERLKA/kg soil, equal to 235 mg/kg pure calcium cyanamide) was made. One month and one year, respectively, earthworms were samples and earthworm abundance, the number of earthworms per species, body weight of all sampled earthworms and body weight of each earthworm species were determined. No statistically significant reduction in earthworm abundance and biomass of earthworms was observed, one month and 12 months after application, respectively.

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-30 to 2016-03-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz; Wiesbaden, 12 June 2015

Specific details on test material used for the study:

Mean concentration of calcium cyanamide in PERLKA is 44 %.

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil: The test item was applied directly into the test vessels which contained untreated LUFA St. 2.2. The granules were randomly distributed within the test vessels by mixing the soil for at least one minute (e.g. with a spatula). All test vessels were loaded by a total amount of granules to achieve the respective concentrations.
- Controls: For the control no granules were added.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: The earthworms used in this test, Eisenia fetida (Lumbricidae, Earthworms) have been kept at ECT Oekotoxikologie GmbH since February 1994. They were originally delivered by Co. Landenberger (D-72355 Schömberg). The earthworms for the test were taken from a synchronised culture.
- Age at test initiation (mean and range, SD): 2 - 12 months, however the age of individuals did not differ by more than four weeks
- Weight at test initiation (mean and range, SD): 250 - 600 mg

ACCLIMATION
- Acclimation period: at least 24 h
- Acclimation conditions (same as test or not): same as test conditions
- Health during acclimation (any mortality observed): not specified

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

Mean 20.0 °C, 18.2 – 21.9 °C

pH:

5.8 - 5.9

Moisture:

20.6 - 20.8 % of dry mass; 47.1 - 47.9 % of maximum water holding capacity

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Test vessels were Bellaplast containers (Company Kastelplast, 55120 Mainz, Germany) made from inert (non-toxic) plastic (Polystyrol). The test vessels have a base area of 11 x 15.5 cm (= 170.5 cm²) and a height of 6 cm. The test vessels were covered with a transparent and perforated lid.
- Amount of soil or substrate: Per test vessel an amount corresponding to 500 g dw (dry weight) was used. The height of the substrate surface was ca. 5 cm
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil: standard soil LUFA St. 2.2
- Source: Purchased from the “Landwirtschaftliche Untersuchungs- und Forschungsanstalt”, 67346 Speyer, Germany.
- Soil texture (if natural soil): loamy sand
- Particle size distribution (USDA):
< 0.002 mm: 7.7 ± 1.7,
0.002 - 0.05 mm: 16.2 ± 2.6,
0.05 – 2.0 mm: 76.1 ± 4.0
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- Organic carbon (%): 1.59 ± 0.13
- Nitrogen [%]: 0.17 ± 0.01
- pH (0.01 M CaCl2): 5.4 ± 0.2
- Maximum water holding capacity (in % dry weigth): 43.5 ± 2.8
- CEC: 9.7 ± 0.4 mval/100 g
- Weight per volume [g/1000 mL]: 1218 ± 47
- Actual water content [% dry weight]: 6.4
- Pretreatment of soil: One day before starting the test the LUFA St. 2.2 soil was pre-moistened (per replicate 500 g dw). Additionally to the actual water content deionised water was added to get a soil moisture of 40 – 60% of WHCmax minus volume of application solution

OTHER TEST CONDITIONS
- Photoperiod: 16:8 h (light:dark)
- Light intensity: 432 - 669 Lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality (28d), Biomass (28d) and Reproduction (56d)

VEHICLE CONTROL PERFORMED: no vehicle used, therefore, a water treated control was performed

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Range finding study: yes, non-GLP, not covered by the study plan
- Test concentrations: 30, 50, 82, 135, 223, 367, 606 and 1000 mg test item/kg soil (dw)

Nominal and measured concentrations:

nominal:
30 mg test item/kg soil (dw)
50 mg test item/kg soil (dw)
82 mg test item/kg soil (dw)
135 mg test item/kg soil (dw)
223 mg test item/kg soil (dw)
367 mg test item/kg soil (dw)
606 mg test item/kg soil (dw)
1000 mg test item/kg soil (dw)

Reference substance (positive control):

yes

Remarks:

Boric acid

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

36.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: The mean concentration of calcium cyanamide in PERLKA is 44 %. Thus, the NOEC for pure calcium cyanamide is 36.08 mg/kg soil dw.

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

82 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

135 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

153.7 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95%-confidence interval: 113.0 – 186.8 mg test item/kg soil (dw)

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

347.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95%-confidence interval: 308.6 – 390.5 mg test item/kg soil (dw)

Duration:

56 d

Dose descriptor:

other: EC20

Effect conc.:

203.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95%-confidence interval: 162.5 – 236.2 mg test item/kg soil (dw)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Remarks on result:

other: highest concentration tested

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

367 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

606 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

720.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: After 28 days 1.25% mortality was observed in the control, 0 - 10% at the concentrations of 30, 50, 82, 135, 223 and 367 mg test item/kg soil (dw). At the concentrations of 606 and 1000 mg test item/kg soil (dw) 37.5% and 85.0% mortality was observed, respectively. Statistical analysis (Fisher’s exact binomial test with Bonferroni correction; 1-sided, p ≤ 0.05) showed a significant difference concerning mortality between the water control and the concentrations of 606 and 1000 mg test item/kg soil (dw). Therefore, the NOEC (Mortality) was determined as 367 mg test item/kg soil (dw) and accordingly the LOEC (Mortality) as 606 mg test item/kg soil (dw). The LC50 for mortality was calculated as 720.9 mg test item/kg soil.
- Total mass of adults at beginning of test: please refer to table 3
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Statistical analysis showed no significant difference (Welch t-test, 2-sided; p ≤ 0.05) for biomass development of individual adults over 28 days between the control and all eight concentrations of the test item (please refer to table 3).
- No. of offspring produced: Statistical analysis (Williams test; 1-sided, p ≤0.05) showed a significant difference concerning the number of juveniles between the control and the concentrations of 135, 223, 367, 606 and 1000 mg test item/kg soil dw. Therefore, the NOEC (Reproduction) was determined as 82 mg test item/kg soil (dw) and accordingly the LOEC (Reproduction) as 135 mg test item/kg soil (dw) (please refer to table 4 and attached figure).
- Other biological observations: The feeding activity was reduced after 7 days in the concentrations of 223, 367, 606 and 1000 mg test item/kg soil (dw). After 14 days a reduced feeding activity was observed in one replicate (D) of the concentration 367 mg test item/kg soil (dw) and the concentrations of 606 and 1000 mg test item/kg soil. 21 and 28 days after test start a reduced feeding activity was observed in the concentrations of 606 and 1000 mg test item/kg soil. At all other test vessels almost the whole food was consumed by the worms after 7, 14, 21 and 28 days of the test start of the test item.
No other effects on behavior or morphology of the adult earthworms were observed

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 259.1 mg boric acid/kg soil (dw) (% confidence limits = 234.0 – 286.2 mg boric Acid/kg artificial soil (dw))
- Other: The estimated EC50 value is slightly lower than recommended by the guideline. However, since the calculated EC50 is lower only by factor of 1.5 than the recommended value of 400 mg boric acid/ kg soil (dw) acceptable sensitivity of the test system is assumed.

Table 1: Effects on Mortality (28d), Biomass (28d) and Reproduction (56d) of Eisenia fetida.

Concentration [mg test item/kg soil (dw)]	Adult mortality [%]	Biomass [Mean change in % of the initial weight per replicate]	Number of Juveniles[% of Control]
Control	1.25	60.0	100.0
30	0.0	64.9	97.0
50	0.0	60.8	109.5
82	10.0	69.1	99.7
135	2.5	76.3	82.1**
223	0.0	74.4	78.0**
367	2.5	64.6	54.5**
606	37.5*	68.5	12.0**
1000	85.0*	83.4	1.1**
NOEC [mg test item/kg soil (dw)]	367	> 1000	82
LOEC [mg test item/kg soil (dw)]	606	> 1000	135
EC10 [mg test item/kg soil (dw)]			153.7
EC20 [mg test item/kg soil (dw)]			203.3
LC50/EC50 [mg test item/kg soil (dw)]	720.9		347.1
* Statistically significant different to control (Fisher exact test, 1-sided; p < 0.05) ** Statistically significant different to solvent control (Williams test, 1-sided; p < 0.05)

Table 2: Number of dead earthworms after 28 days (n = 80 for control; n = 40 for concentrations). Data are given as absolute values and in percent of the total number (mortality).

Concentration [mg test item/kg soil (dw)]	Number of dead adult worms	Mortality [%]
	Day 28	Day 28
Control	1	1.25
30	0	0.0
50	0	0.0
82	4	10.0
135	1	2.5
223	0	0.0
367	1	2.5
606	15	37.5*
1000	34	85.0*
* Statistically significant different to control (Fisher exact test, 1-sided; p < 0.05)

Table 3: Average live weight (mg) per surviving adult earthworm at the beginning of the test and after 28 days (n = 80 for control; n = 40 for concentrations). Data are given as absolute value and in mean percent of the initial weight change per replicate.

Concentration [mg test item/kg soil (dw)]	Biomass (mg fresh weight)		Mean change in % of the initial weight per replicate
	Day0	Day28	Day 28
Control	342.6±24.2	546.4±36.7	60.0
30	338.2±24.0	555.1±16.5	64.9
50	338.1 ± 23.7	541.7±15.8	60.8
82	338.4± 22.8	570.7 ± 30.2	69.1
135	337.9±21.0	594.3 ± 7.8	76.3
223	338.4±19.4	589.1 ± 17.2	74.4
367	340.2±15.4	558.7 ± 33.4	64.6
606	339.7±14.8	572.2 ± 48.0	68.5
1000	341.1 ±11.8	624.7±116.4	83.4

Table 4: Reproduction (given as mean number of juveniles ± standard deviation and as % of the control) in the treated test vessels.

Concentration [mg test item/kg soil (dw)]	Number of Juveniles per Treatment (mean ± sd)	Number of Juveniles [% of Control]
Control	371.1 ± 59.9	100.0
30	359.8 ± 68.3	97.0
50	406.3 ± 22.1	109.5
82	370.0 ± 63.1	99.7
135	304.8 ± 12.4	82.1*
223	289.3 ± 43.8	78.0*
367	202.3 ± 37.8	54.5*
606	44.5 ± 50.1	12.0*
1000	4.0 ± 4.9	1.1*
* Statistically significant different to control (Williams test, 1-sided; p < 0.05)

Validity criteria fulfilled:

yes

Conclusions:

The study results are to be multiplied by 0.44 to calculate from the given concentration of the relevant active ingrenident calcium cyanamide in PERLKA (R) of 44% to 100% of calcium cyanamide
NOEC (Mortality) = 367 mg test item/kg soil (dw). (eqivalent to 161.5 mg Calcium Cyanamide)
LOEC (Mortality) = 606 mg test item/kg soil (dw). (eqivalent to 266.6 mg Calcium Cyanamide)
LC50 (Mortality) = 720.9 mg test item/kg soil (dw) (eqivalent to 317.2 mg Calcium Cyanamide)
NOEC (Biomass) ≥ 1000 mg test item/kg soil (dw). (eqivalent to > 440 mg Calcium Cyanamide)
LOEC (Biomass) > 1000 mg test item/kg soil (dw). (eqivalent to > 440 mg Calcium Cyanamide)
NOEC (Reproduction) = 82 mg test item/kg soil (dw). (eqivalent to 36.1 mg Calcium Cyanamide)
LOEC (Reproduction) = 135 mg test item/kg soil (dw). (eqivalent to 59.4 mg Calcium Cyanamide)
EC10 (Reproduction) = 153.7 mg test item/kg soil (dw). (eqivalent to 67.6 mg Calcium Cyanamide)
EC20 (Reproduction) = 203.3 mg test item/kg soil (dw). (eqivalent to 89.5 mg Calcium Cyanamide)
EC50 (Reproduction) = 347.1 mg test item/kg soil (dw). (eqivalent to 152.7 mg Calcium Cyanamide)

Executive summary:

A study according to OECD guideline 222 (Earthworm Reproduction Test (Eisenia fetida/ Eisenia andrei)) was conducted with the Calcium Cyanamide in the form of its fertiliser mixture "PERLKA". The purpose of this study was to determine a NOEC/LOEC and if possible, an EC10, EC20 and EC50 value for reproduction (56 days after application) and a NOEC/LOEC value for mortality and the biomass development (28 days after application) of the earthworm Eisenia fetida (Lumbricidae) using a standard LUFA 2.2 soil. The test item was mixed with the substrate. The earthworm Eisenia fetida (Lumbricidae) was chosen as a representative of the soil fauna.

Biomass was not affected by the treatment. On the other hand, an increased of mortality after 28 days was observed (37.5% and 85.0% at the concentrations of 606 and 1000 mg test item/kg soil (dw) equiv. to 976.3 mg and 1611 mg Calcium Cyanamide, respectively). In addition, the reproduction was reduced at concentrations starting at 135 mg test item/kg soil (dw). The NOEC (reproduction) after 56 days was 82 mg PERLKA/kg soil dw, equiv. to 36.1 mg calcium cyananamide/kg soil dw.

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

not specified

GLP compliance:

not specified

Analytical monitoring:

not specified

Details on sampling:

no data

Vehicle:

not specified

Details on preparation and application of test substrate:

no data

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

clitellated adult earthworms

Study type:

laboratory study

Substrate type:

not specified

Limit test:

no

Total exposure duration:

14 d

Post exposure observation period:

not indicated

Test temperature:

not indicated

pH:

not indicated

Moisture:

not indicated

Details on test conditions:

Three replicates of 6 clitellated adult earthworms were each exposed to five doses in a geometric series, in order to determine a LC50 value. Controls were replicated 3 times, with 6 earthworms in each replicate. No toxic reference standard was tested. Worms were assessed for mortality and sublethal effects after 7 and 14 days of exposure and earthworm body weights were assessed at day 0 and day 14.

Nominal and measured concentrations:

42.2% Calcium cyanamide

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

261 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: based on test substance as received

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

110.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: re-calculation of result for PERLKA to 100 % calcium cyanamide

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

261.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: result for product PERLKA with 42.2 % calcium cyanamide

Details on results:

The LC50 of PERLKA was determined to be 261.4 mg product/kg dry soil, with a confidence interval of 251.3 to 272.0 mg product/kg dry soil, and a slope of 95%. This corresponds to 110.3 mg/kg soil dw for pure calcium cyanamide.

Results with reference substance (positive control):

not indicated

Reported statistics and error estimates:

not indicated

Validity criteria fulfilled:

not specified

Conclusions:

The acute earthworm LC50 of calcium cyanamide was determined to be 261.4 mg product/kg dry soil, equivalent to 110.3 mg active ingredient/kg dry soil.

Executive summary:

Acute toxicity of PERLKA to earthworms, Eisenia foetida Michaelsen, was determined in a 14-day soil exposure laboratory study conducted similar to OECD Guideline 207. PERLKA contains 42.2% calcium cyanamide.

Three replicates of 6 clitellated adult earthworms were each exposed to five doses in a geometric series, in order to determine a LC50 value. Controls were replicated 3 times, with 6 earthworms in each replicate. No toxic reference standard was tested. Worms were assessed for mortality and sublethal effects after 7 and 14 days of exposure and earthworm body weights were assessed at day 0 and day 14.

The LC50 of PERLKA was determined to be 261.4 mg product/kg dry soil, with a confidence interval of 251.3 to 272.0 mg product/kg dry soil, and a slope of 95%. This corresponds to 110.3 mg/kg soil dw for pure calcium cyanamide.

Reference 5

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Justification for read-across for ecotoxicological endpoints:
Environmental fate and exposure are critical factors when assessing environmental risk. Calcium cyanamide is formulated in a slowly dissolving granule (PERLKA) that is applied to agricultural fields as a fertiliser. When dissolved, calcium cyanamide is rapidly converted to (hydrogen) cyanamide. These two substances are similar both in chemical structure and regarding fate in the environment. Cyanamide is the environmentally relevant transformation product upon application of calcium cyanamide (PERLKA) to soil as a fertiliser. Therefore, terrestrial data on cyanamide are considered in the hazard assessment of calcium cyanamide.
For further details please refer to the read-across report for ecotoxicological endpoints in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.05 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: highest concentration tested

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 1.05 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: highest concentration tested

Key result

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1.05 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: highest concentration tested

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.05 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: biomass

Remarks on result:

other: highest concentration tested

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1.05 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: biomass

Remarks on result:

other: highest concentration tested

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.05 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: highest concentration tested

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1.05 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: highest concentration tested

Details on results:

- Mortality at end of exposure period: 6.3% mortality was observed in the control. In all concentrations of the test item tested 0 to 12.5% mortality was observed. Fisher’s Exact Test (1-sided, p ≤ 0.05) showed no significant differences concerning mortality between the control and all test item concentrations. Therefore, NOEC (mortality) was considered to be ≥ 1.05 mg a.s./kg soil (dw) (the highest concentration of the test item). The LOEC (mortality) could not be determined and was regarded as > 1.05 mg a.s./kg soil (dw).
- Total mass of adults at beginning of test: please refer to table 3
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Statistical analysis showed no significant difference (Dunnett’s test, 2-sided; p ≤ 0.05) for biomass development of individual adults over 28 days between the control and all eight concentrations of the test item. Therefore, the NOEC (Biomass) was considered to be ≥ 1.05 mg a.s./kg soil (dw) (the highest concentration of the test item). The LOEC (Biomass) could not be determined and was regarded as > 1.05 mg a.s./kg soil (dw) (please refer to table 3).
- No. of offspring produced: Statistical analysis (Dunnett’s test; 1-sided, p ≤ 0.05) showed no significant difference concerning the number of juveniles between the control and all concentrations tested. Therefore, the NOEC (Reproduction) was considered to be ≥ 1.05 mg a.s./kg soil (dw) and the LOEC (Reproduction) was regarded as > 1.05 mg a.s./kg soil (dw). Since inhibition of reproduction was < 50%, the EC50 could not be calculated and was considered to be > 1.05 mg a.s./kg soil (dw) (please refer to table 4 and attached figure).
- Morphological abnormalities: No effects on morphology of the adult earthworms were observed.
- Behavioural abnormalities: No effects on behavior of the adult earthworms were observed.
- Other biological observations: The feeding activity was reduced in the concentration of 0.017 mg a.s./kg soil (dw) at replicate A (21 and 28 days after application) and replicate D (28 days after application), in replicate D of the concentration of 0.056 mg a.s./kg soil (dw) (21 and 28 days after application), in replicates A (14, 21 and 28 days after application) and C (7, 14 and 28 days after application) of the concentration of 0.10 mg a.s./kg soil (dw) and in replicates C (14, 21 and 28 days after application) and D (28 days after application) of the concentration of 0.32 mg a.s./kg soil (dw). At all other test vessels almost the whole food was consumed by the worms after 7, 14, 21 and 28 days of the application of the test item.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 259.1 mg boric acid/kg soil (dw) (% confidence limits = 234.0 – 286.2 mg boric Acid/kg artificial soil (dw))
- Other: The estimated EC50 value is slightly lower than recommended by the guideline. However, since the calculated EC50 is lower only by factor of 1.5 than the recommended value of 400 mg boric acid/ kg soil (dw) acceptable sensitivity of the test system is assumed.

Table 1: Effects on Mortality (28d), Biomass (28d) and Reproduction (56d) of Eisenia fetida.

Concentration [mg a.s./kg soil (dw)]	Adult mortality [%]	Biomass [Mean change in % of the initial weight per replicate]	Number of Juveniles [% of Control]
Control	6.3	39.2	100.0
0.017	10.0	36.4	70.9
0.031	2.5	38.7	80.9
0.056	5.0	38.3	74.8
0.10	12.5	36.8	72.3
0.18	2.5	42.9	97.5
0.32	5.0	38.0	85.3
0.58	7.5	41.3	94.8
1.05	0.0	45.5	103.4
NOEC [mg a.s./kg soil (dw)]	≥ 1.05	≥ 1.05	≥ 1.05
LOEC [mg a.s./kg soil (dw)]	> 1.05	> 1.05	> 1.05
EC50 [mg a.s./kg soil (dw)]			> 1.05

Table 2: Number of dead earthworms after 28 days (n = 80 for control; n = 40 for concentrations). Data are given as absolute values and in percent of the total number (mortality).

Concentration [mg a.s./kg soil (dw)]	Number of dead adult worms	Mortality [%]
	Day 28	Day 28
Control	5	6.3
0.017	4	10.0
0.031	1	2.5
0.056	2	5.0
0.10	5	12.5
0.18	1	2.5
0.32	2	5.0
0.58	3	7.5
1.05	0	0.0

Table 3: Average live weight (mg) per surviving adult earthworm at the beginning of the test and after 28 days (n = 80 for control; n = 40 for concentrations). Data are given as absolute value and in mean percent of the initial weight change per replicate.

Concentration [mg a.s./kg soil (dw)]	Biomass (mg fresh weight)		Mean change in % of the initial weight per replicate
	Day 0	Day 28	Day 28
Control	438.6 ± 29.5	609.5 ± 43.5	39.2
0.017	438.7 ± 35.8	595.8 ± 11.3	36.4
0.031	438.9 ± 33.0	608.1 ± 30.6	38.7
0.056	439.3 ± 32.4	605.9 ± 40.5	38.3
0.10	439.6 ± 29.9	598.7 ± 51.7	36.8
0.18	439.9 ± 27.2	628.1 ± 34.2	42.9
0.32	440.6 ± 27.1	606.4 ± 23.2	38.0
0.58	439.7 ± 25.7	619.0 ± 48.6	41.3
1.05	440.3 ± 21.4	638.6 ± 27.2	45.5

Table 4: Reproduction (given as mean number of juveniles ± standard deviation and as % of the control) in the treated test vessels.

Concentration [mg a.s./kg soil (dw)]	Number of Juveniles per Treatment (mean ± sd)	Number of Juveniles [% of Control]
Control	425.5 ± 88.9	100.0
0.017	301.5 ± 123.0	70.9
0.031	344.3 ± 77.8	80.9
0.056	318.3 ± 150.4	74.8
0.10	307.5 ± 88.7	72.3
0.18	414.8 ± 49.1	97.5
0.32	362.8 ± 160.0	85.3
0.58	403.3 ± 81.7	94.8
1.05	439.8 ± 41.0	103.4

Validity criteria fulfilled:

yes

Conclusions:

NOEC (Mortality) ≥1.05 mg a.s./kg soil (dw).
LOEC (Mortality) >1.05 mg a.s./kg soil (dw).
NOEC (Biomass) ≥1.05 mg a.s./kg soil (dw).
LOEC (Biomass) >1.05 mg a.s./kg soil (dw).
NOEC (Reproduction) ≥1.05 mg a.s./kg soil (dw).
LOEC (Reproduction) >1.05 mg a.s./kg soil (dw).
EC50 >1.05 mg a.s./kg soil (dw).

Justification for read-across for ecotoxicological endpoints:
Environmental fate and exposure are critical factors when assessing environmental risk. Calcium cyanamide is formulated in a slowly dissolving granule (PERLKA) that is applied to agricultural fields as a fertiliser. When dissolved, calcium cyanamide is rapidly converted to (hydrogen) cyanamide. These two substances are similar both in chemical structure and regarding fate in the environment. Cyanamide is the environmentally relevant transformation product upon application of calcium cyanamide (PERLKA) to soil as a fertiliser. Therefore, terrestrial data on cyanamide are considered in the hazard assessment of calcium cyanamide.
For further details please refer to the read-across report for environmental endpoints in IUCLID section 13.

Executive summary:

A study was conducted followintg the the internationally accepted OECD test guideline 222 (Earthworm Reproduction Test (Eisenia fetida/ Eisenia andrei)) in accordance with GLP.

The study was conducted with a 50% aqueous solution of Cyanamide. The purpose of this study was to determine a NOEC/LOEC and if possible, an EC10, EC20 and EC50 value for reproduction (56 days after application) and a NOEC/LOEC value for mortality and the biomass development (28 days after application) of the earthworm Eisenia fetida (Lumbricidae) using a standard LUFA 2.2 soil. The test item was mixed with the substrate. The earthworm Eisenia fetida (Lumbricidae) was chosen as a representative of the soil fauna.

Biomass, mortality and reproduction were not affected by the treatment. Therefore, the NOEC for biomass (28 d), mortality (28 d) and reproduction (56 d) is ≥1.05 mg a.s./kg soil dw.

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

For justification of read-across for ecotoxicological endpoints please refer to the section "Conclusion".

Reference 6

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

Justification for read-across for ecotoxicological endpoints:
Environmental fate and exposure are critical factors when assessing environmental risk. Calcium cyanamide is formulated in a slowly dissolving granule (PERLKA) that is applied to agricultural fields as a fertiliser. When dissolved, calcium cyanamide is rapidly converted to (hydrogen) cyanamide. These two substances are similar both in chemical structure and regarding fate in the environment. Cyanamide is the environmentally relevant transformation product upon application of calcium cyanamide (PERLKA) to soil as a fertiliser. Therefore, terrestrial data on cyanamide are considered in the hazard assessment of calcium cyanamide.
For further details please refer to the read-across report for ecotoxicological endpoints in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 111.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: corresponding to 1000 mg/kg soil dw of test material

Details on results:

No significant sublethal behavioural effects were observed up to a rate of 500 mg SKW Cyanamide L500 + AHL/kg dry soil weight.
The NOEC was determined to be 500 mg SKW Cyanamide L500 + AHL /kg dry soil weight. A statistically significant loss of body weight equivalent to 69.7% was observed in the 1000 mg SKW cyanamide L500 + AHL/kg soil dry weight test concentration.
The LC50 for the toxic reference standard was 21.1 mg/kg soil dry weight; this is within accepted limits indicating the validity of this test.

Results with reference substance (positive control):

The LC50 for the toxic reference standard was 21.1 mg/kg soil dry weight; this is within accepted limits indicating the validity of this test.

Reported statistics and error estimates:

The test group results were significantly different compared to the control by the Dunnett test, α=0.05

Acute toxicity of SKW Cyanamid L500 + AHL to earthworms:

Treatment	Cumulative Mortality (%)^a		Cumulative Weight Loss (%)^b
(mg SKW Cyanamid L500 + AHL/kg dry soil)	7 days	14 days	14 days
0	0	0	-
62.5	0	0	3.4
125	0	0	33.1
250	0	0	26.2
500	0	2.5	12.4
1000	0	0	-69.7*

Validity criteria fulfilled:

yes

Conclusions:

The acute earthworm LC50 of SKW Cyanamid L500 + AHL was determined to be >1000 mg/kg dry soil weight (equivalent to >111.3 mg as/kg dry soil) the highest rate tested.

Justification for read-across for ecotoxicological endpoints:
Environmental fate and exposure are critical factors when assessing environmental risk. Calcium cyanamide is formulated in a slowly dissolving granule (PERLKA) that is applied to agricultural fields as a fertiliser. When dissolved, calcium cyanamide is rapidly converted to (hydrogen) cyanamide. These two substances are similar both in chemical structure and regarding fate in the environment. Cyanamide is the environmentally relevant transformation product upon application of calcium cyanamide (PERLKA) to soil as a fertiliser. Therefore, terrestrial data on cyanamide are considered in the hazard assessment of calcium cyanamide.
For further details please refer to the read-across report for environmental endpoints in IUCLID section 13.

Executive summary:

The acute toxicity of SKW Cyanamid L500 + AHL to earthworms, Eisenia fetida, was determined in a 14-day soil exposure laboratory study. Four replicates of 10 clitellated adult earthworms were each exposed to nominal concentrations of 62.5, 125, 250, 500 and 1000 mg SKW Cyanamid L500 + AHL/kg dry soil weight (ppm in dry soil). Controls were replicated 4 times, with 10 earthworms in each replicate. The toxic reference standard was tested at 5 rates in a separate study. Worms were assessed for mortality and sublethal effects after 7 and 14 days of exposure and earthworm body weights were assessed at day 0 and day 14. No significant sublethal behavioural effects were observed up to a rate of 500 mg SKW Cyanamid L500 + AHL/kg dry soil weight. At an application rate of 1000 mg SKW Cyanamid L500 + AHL /kg dry soil weight, a significant cumulative weight loss of 69.7 % was observed. The LC50 for the toxic reference standard was 21.1 mg/kg soil dry weight; this is within accepted limits indicating the validity of this test.

It can be concluded that the acute earthworm LC50 of SKW Cyanamid L500 + AHL was determined to be >1000 mg/kg dry soil (equivalent to >111.3 mg active substance/kg dry soil), the highest rate tested.

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

For justification of read-across for ecotoxicological endpoints please refer to the section "Conclusion".

Description of key information

The most sensitive acute LC50 of 261 mg PERLKA/kg dry soil (equivalent to 110.3 mg calcium cyanamide/kg soil dw and 57.97 mg cyanamide/kg soil dw, respectively) for terrestrial macroorganisms (Eisenia foetida Michaelsen) is reported by Haque & Ebing (1983).

The 56-d NOEC for reproduction of Eisenia foetida is 82 mg PERLKA/kg soil dry weight (equivalent to 36.1 mg calcium cyanamide/kg soil dw and 18.97 mg cyanamide/kg soil dw, respectively) reported by Scheffczyk (2016a).

Information on the transformation product cyanamide is as well available and considered in the terrestrial hazard assessment in a read-across approach:

Lührs (2001) tested the acute toxicity effects of cyanamide on earthworms in a 14-day laboratory study. The LC50 observed after 14 days of incubation was  > 111.3 mg/kg dry soil (active substance cyanamide), the highest rate tested.

Further investigations on the chronic toxicity of cyanamide to Esenia fetida are reported by Scheffczyk (2016b). The NOEC for reproduction is ≥ 1.05 mg cyanamide/kg soil dw, the highest concentration investigated during the study.

For justification of read-across for environmental endpoints please refer to the document in IUCLID section 13.

The study conducted by Scheffczyk (2016b) investigates the 56-d reproduction toxicity of cyanamide in earthworm (Eisenia fetida) in accordance with OECD guideline 222 and ISO 11268-2.

In the first step a 28-d range-finding study (non-GLP) was conducted at 0 (control), 0.1, 1, 10, 100 and 100 mg cyanamide/kg soil (dw) in two replicates. Based on the dose-response relationship observed in the range-finding study, the concentration range for the definitive test (GLP) was chosen as follows: 0 (control), 0.017, 0.031, 0.056, 0.10 mg, 0.18, 0.32, 0.58, and 1.05 mg cyanamide/kg soil (dw).

 

In the definitive test, no adverse effects could be observed at the highest concentration evaluated in the test. Thus, the NOEC from this study is ≥ 1.05 mg cyanamide/kg soil dw, a definitive LOEC value could not be derived.

Although the validity criteria of OECD guideline 222 are met (mortality in control ≤ 10 %; number of juveniles per control replicate ≥ 30; coefficient of variation for the number of juveniles in control ≤ 30 %), the variability between replicates in the control and in samples of all test concentrations is relatively high, thus reducing the robustness of the data.

 

In accordance with the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.10: Characterisation of dose [concentration]-response for environment (May 2008, p. 11), the NOEC is defined as “the highest concentration tested at which the substance is observed to have no statistically significant effect (p<0.05) when compared with the control, within a stated exposure period” (OECD 211, 1998b) or the test concentration immediately below the LOEC, which when compared with the control has no statistically significant effect (p<0.05) within a stated period (OECD 211, 1998b).” Furthermore, “[t]here has to be a concentration-effect relationship.”

However, in the present main test by Scheffczyk (2016b), no concentration-effect relationship was observed.

 

Any bounded NOEC of cyanamide for the reproduction of E. fetida under the conditions of OECD guideline 222 would presumably be higher than the highest test concentration chosen is the test by Scheffczyk (2016b). However, such a bounded NOEC value is currently not available and could only be determined by repeat testing.

 

Besides, preliminary information from the agricultural field study (Ebke, 2018; see IUCLID section 6.6) indicate no adverse effects on populations of earthworms at a single annual application rate of 400 kg PERLKA/ha that corresponds to a concentration of 11.8 mg cyanamide/kg soil (dw) (see ESCAPE V2 calculations in IUCLID section 13.2; Kiefer, 2018). This concentration is considerably higher compared to the highest test concentration applied in the study by Scheffczyk (2016b).

 

In conclusion, the unbounded NOEC of ≥ 1.05 mg cyanamide/kg soil (dw) in E. fetida reported byScheffcyzk (2016b) is not considered relevant for hazard assessment purposes.

As no specific LC50 and NOEC values are available for cyanamide, the information generated for calcium cyanamide is carried forward in the chemical safety assessment.

As cyanamide is the environmentally relevant transformation product of calcium cyanamide, effect values are re-calculated to cyanamide on the basis of molar masses for both substances.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

57.97 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

18.97 mg/kg soil dw

Additional information

The key study was conducted according to OECD guideline 222 (Earthworm Reproduction Test (Eisenia fetida/ Eisenia andrei)) with calcium cyanamide in the form of its fertiliser mixture "PERLKA" (Scheffczyk, 2016a). The purpose of this study was to determine a NOEC/LOEC and if possible, an EC10, EC20 and EC50 value for reproduction (56 days after application) and a NOEC/LOEC value for mortality and the biomass development (28 days after application) of the earthworm Eisenia fetida (Lumbricidae) using a standard LUFA 2.2 soil. The test item was mixed with the substrate. The earthworm Eisenia fetida (Lumbricidae) was chosen as a representative of the soil fauna.

Biomass was not affected by the treatment. On the other hand, an increased of mortality after 28 days was observed (37.5% and 85.0% at the concentrations of 606 and 1000 mg test item/kg soil (dw) equiv. to 976.3 mg and 1611 mg Calcium Cyanamide, respectively). In addition, the reproduction was reduced at concentrations starting at 135 mg test item/kg soil (dw). The NOEC (reproduction) after 56 days was 82 mg PERLKA/kg soil dw, equiv. to 36.1 mg calcium cyananamide/kg soil dw (corresponding to 18.97 mg cyanamide/kg soil dw).

This information is supplemented by acute toxicity studies:

Acute toxicity of calcium cyanamide to earthworms, Eisenia foetida and Lumbricus terrestris, was determined in a 14-day soil exposure laboratory study conducted similar to OECD Guideline 207 (Heimbach, 1984). Three replicates of 6 or 10 clitellated adult earthworms were each exposed to doses in a geometric series, in order to determine a LC50 value. No toxic reference standard was tested. Worms were assessed for mortality and sublethal effects after 7 and 14 days of exposure.

Mortality results and sublethal effects were not reported for each concentration. The acute earthworm LC50 of calcium cyanamide was determined to be 115 mg calcium cyanamide/kg dry soil for Eisenia foetida and 124 mg calcium cyanamide/kg dry soil for Lumbricus terrestris.

In a second study, the acute toxicity of PERLKA to earthworms (Eisenia foetida Michaelsen) was determined in a 14-day soil exposure laboratory study conducted similar to OECD Guideline 207 (Haque & Ebing, 1983). PERLKA contains 42.2% calcium cyanamide. Three replicates of 6 clitellated adult earthworms were each exposed to five doses in a geometric series, in order to determine a LC50 value. Controls were replicated 3 times, with 6 earthworms in each replicate. No toxic reference standard was tested. Worms were assessed for mortality and sublethal effects after 7 and 14 days of exposure and earthworm body weights were assessed at day 0 and day 14.

The LC50 of PERLKA was determined to be 261.4 mg product/kg dry soil, with a confidence interval of 251.3 to 272.0 mg product/kg dry soil, and a slope of 95%. The LC50 corresponds to 110.3 mg/kg soil dw for pure calcium cyanamide (= 57.97 mg cyanamide/kg soil dw).

 

In a supporting field study on grassland (meadow) PERLKA was tested for possible side effects on earthworms (Bauchhenß, 1992). A single broadcast application at a rate of 400 kg PERLKA/ha (533 mg PERLKA/kg soil, equal to 213.2-239.85 mg/kg pure calcium cyanamide) was made. One month and one year, respectively, earthworms were sampled and earthworm abundance, the number of earthworms per species, body weight of all sampled earthworms and body weight of each earthworm species were determined. No statistically significant reduction in earthworm abundance and biomass of earthworms was observed, one month and 12 months after application, respectively.

In addition, information on the transformation product cyanamide is considered in the hazard assessment:

The acute toxicity of cyanamide (SKW Cyanamid L500 + AHL (synonymous to urea ammonium nitrate)) to earthworms, Eisenia fetida, was determined in a 14-day soil exposure laboratory study (Lührs, 2001). Four replicates of 10 clitellated adult earthworms were each exposed to nominal concentrations of 62.5, 125, 250, 500 and 1000 mg SKW Cyanamid L500 + AHL/kg dry soil weight (ppm in dry soil). Controls were replicated 4 times, with 10 earthworms in each replicate. The toxic reference standard was tested at 5 rates in a separate study. Worms were assessed for mortality and sublethal effects after 7 and 14 days of exposure and earthworm body weights were assessed at day 0 and day 14. No significant sublethal behavioural effects were observed up to a rate of 500 mg SKW Cyanamid L500 + AHL/kg dry soil weight. At an application rate of 1000 mg SKW Cyanamid L500 + AHL /kg dry soil weight, a significant cumulative weight loss of 69.7 % was observed. It can be concluded that the LC50 for the toxic reference standard was 21.1 mg/kg soil dry weight; this is within accepted limits indicating the validity of this test.

It can be concluded that the acute earthworm LC50 of SKW Cyanamid L500 + AHL was determined to be > 111.3 mg/kg dry soil (active substance cyanamide), the highest rate tested.

Scheffczyk (2016) conducted a second study according to OECD guideline 222 (Earthworm Reproduction Test (Eisenia fetida/ Eisenia andrei)) with a 50% aqueous solution of cyanamide. The purpose of this study was to determine a NOEC/LOEC and if possible, an EC10, EC20 and EC50 value for reproduction (56 days after application) and a NOEC/LOEC value for mortality and the biomass development (28 days after application) of the earthworm Eisenia fetida (Lumbricidae) using a standard LUFA 2.2 soil. The test item was mixed with the substrate. The earthworm Eisenia fetida (Lumbricidae) was chosen as a representative of the soil fauna.

Biomass, mortality and reproduction were not affected by the treatment. Therefore, the NOEC for biomass (28 d), mortality (28 d) and reproduction (56 d) is ≥ 1.05 mg a.s./kg soil (dw).