Calcium cyanamide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-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

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.41 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 0.104 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

immobilisation

Details on results:

Survival of Daphnia magna was not significantly affected in any test level when compared to the control. The day 21 EC50 based on immobility was > 0.41 mg cyanamide/L (based on mean measured test concentrations).
The 0.023 and 0.41 mg/L test levels were significantly different when compared to the control for adult length. The length was not significantly different in the other concentrations compared to the control. The data for the 0.023 mg/L test level were considered aberrant because they did not follow a normal dose-response pattern. Also, the 0.023 mg/L test level had the second highest number of young/adult/reproduction day of all the test levels and control. The significant difference at the 0.41 mg/L test level is considered a test material effect.
The mean weights were not significantly different from the control at any test concentration. The time to first brood was 7 days for the control and all test concentrations. Thus, it can be concluded that the time to first brood is not influenced by cyanamide.
The 0.41 mg/L test level was significantly different when compared to the control for the reproduction mean. The remaining levels were not significantly different from the control. There were no sublethal (behavioural) effects noted at any test concentration. Based on the effects on length and reproduction mean at the 0.41 mg/L test level, the LOEC can be identified as 0.21 mg/L (based on mean measured test concentrations). Based on the lack of weight, length, survival and reproduction effects, the 0.1 mg/L test level can be identified as NOEC (>0.1 mg/L based on mean measured test concentrations).

Results with reference substance (positive control):

No positive control

Reported statistics and error estimates:

The test groups were significantly different from control according to the Dunnett test, P < 0.05

Summary of test endpoints following exposure of Daphnia magnato to cyanamide for 21 days:

Mean measured concentrations of cyanamide (mg /L)	Mean adult survival^a (%)	Mean first day of reproduction^b	Mean total alive young^c	Mean adult length (mm)	Mean adult dry weight (mg)
Water Control	100.0	7.0	10.83	4.74	0.97
0.023	100.0	7.0	10.91	4.53*	0.88
0.049	97.5	7.0	11.50	4.63	0.91
0.10	95.0	7.0	10.49	4.68	0.97
0.21	100.0	7.0	9.64	4.61	0.98
0.41	97.5	7.0	8.54*	4.53*	0.92

^a Percent of adult daphnids alive at the end of the test (immobility was synonymous with death)

^b First day that reproduction was observed in the replicates

^c Mean of live young produced per surviving female per reproduction day

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

 

Chronic toxicity (21-d) of cyanamide to daphnids – Summary of endpoints:

Endpoints	Mean measured concentration of cyanamide
EC50(immobility)	> 0.41 mg/L
No observed effect concentration (NOEC)	>0.1 mg/L

 

Validity criteria fulfilled:

yes

Remarks:

see above in "principles of methods if other than guideline". The deviations are considered to have no impact on the validity of the study.

Conclusions:

The 21 day EC50 based on immobility was > 0.41 mg cyanamide/L. The 21 day NOEC (no-observed effect concentration), based on mean measured concentrations and the parameters ”young per adult”, ”reproduction day” and ”length” was >0.1 mg cyanamide/L, for Daphnia magna neonates exposed for 21 days under flow-through conditions.

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 effects of of cyanamide on the growth and reproduction of Daphnia magna (< 24-hours old) were assessed in an unaerated, flow-through, 21-day test. Treatments consisted of a water control and nominal concentrations of 0.025, 0.05, 0.1, 0.2 and 0.4 mg cyanamide/L. Forty daphnids (4 replicates contain 10 daphnids) were exposed to each test level.

All chemical and physical parameters (dissolved oxygen concentration, temperature) in the definitive test were within expected ranges. Mean measured concentrations of cyanamide were 0.023, 0.049, 0.1, 0.21 and 0.41 mg /L and ranged from 92 to 105 % of nominal concentrations. In the test medium the test item was sufficiently stable during the test period of 21 days.

Survival of Daphnia magna was not significantly affected in any test level when compared to the control. The day 21 EC50 based on immobility was > 0.41 mg cyanamide/L (based on mean measured test concentrations).

The 0.023 and 0.41 mg/L test levels were significantly different when compared to the control for adult length. The length was not significantly different in the other concentrations compared to the control. The data for the 0.023 mg/L test level were considered aberrant because they did not follow a normal dose-response pattern. Also, the 0.023 mg/L test level had the second highest number of young/adult/reproduction day of all the test levels and control. The significant difference at the 0.41 mg/L test level is considered a test material effect.

The mean weights were not significantly different from the control at any test concentration. The time to first brood was 7 days for the control and all test concentrations. Thus, it can be concluded that the time to first brood is not influenced by cyanamide.

The 0.41 mg/L test level was significantly different when compared to the control for the reproduction mean. The remaining levels were not significantly different from the control. There were no sublethal (behavioural) effects noted at any test concentration. Based on the effects on length and reproduction mean at the 0.41 mg/L test level, the EC50 can be identified as 0.41 mg/L (based on mean measured test concentrations). Based on the lack of weight, length, survival and reproduction effects, the >0.1 mg/L test level can be identified as NOEC.

This information is used in a read-across approach in the assessment of the target substance. For detailled description where read across is used/recommended and where it is preferrable to refain from read across, please see section 13.2 "read across justification for environmental endpoints" and "Scientific rationale for not using cyanamide as read-across substance for calcium cyanamide on toxicological endpoints"

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Remarks on result:

other: Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Remarks on result:

other: Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Intrinsic rate of population increase

Remarks on result:

other: Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Intrinsic rate of population increase

Remarks on result:

other: Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: age at first brood

Remarks on result:

other: The NOEC for the endpoint age of parents at first brood is set to 0.407 mg/L by expert judgement in the report regarding juvenile daphnids. Statistical evaluation of the data shows a significant effect at 0.407 mg/L, resulting in a NOEC of 0.218 mg/L.

Remarks:

Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: age at first brood

Remarks on result:

other: The LOEC for the endpoint age at first brood is set to be higher than 0.407 mg/L by expert judgement in the report regarding juvenile daphnids. Statistical evaluation of the data shows a significant effect at 0.407 mg/L, resulting in a LOEC of 0.470 mg/L

Remarks:

Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Developmental rate

Remarks on result:

other: The NOEC for developmental rate is set to 0.407 mg/L by expert judgement in the report regarding juvenile daphnids. Statistical evaluation of the data shows a significant effect at 0.407 mg/L, resulting in a NOEC of 0.218 mg/L for juvenile daphnids.

Remarks:

Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Developmental rate

Remarks on result:

other: The LOEC for developmental rate is set to be higher than 0.407 mg/L by expert judgement in the report. Statistical evaluation of the data shows a significant effect at 0.407 mg/L, resulting in a LOEC of 0.470 mg/L for juvenile daphnids.

Remarks:

Valid for daphnids introduced as juveniles and daphnids introduced at adults.

Details on results:

- Behavioural abnormalities:
1) Results regarding daphnids introduced as juveniles (<24h old)
Daphnids showed no signs of discoloration or abnormalities in their behavior. No other clinical signs were observed in any replicate at any concentration tested. Neither any physical nor pathological symptoms were obtained.
2) Results regarding daphnids introduced as adults (7 days old)
Daphnids showed no signs of discoloration or abnormalities in their behavior. No other clinical signs were observed in any replicate at any concentration tested. Neither any physical nor pathological symptoms were obtained.

- Observations on body length:
1) Results regarding daphnids introduced as juveniles (<24h old)
Statistical evaluations revealed a significant effect on length for this concentration. However, only low inhibitions of 6.9 % at the lowest concentration of 0.026 mg Cyanamide/L, and 5.4 % at a concentration of 0.218 mg Cyanamide/L were observed (cf. Table 3).
2) Results regarding daphnids introduced as adults (7 days old)
Statistical evaluations revealed a significant effect on length at concentrations of at concentrations of 0.053 and 0.107 mg Cyanamide/L (8.2 % and 6.6 %, respectively). Higher concentrations did not cause an effect on the length (cf. Table 4).

- Immobilisation
1) Results regarding daphnids introduced as juveniles (<24h old)
At the lowest concentration of 0.026 mg/L 20 % immobility and at the concentrations of 0.053, 0.107 and 0.218 mg/L 10 % immobility was observed. In contrast, no immobility was observed in the control and in the highest treatment of 0.470 mg/L. There was no significant difference in survival of individuals between the control and any treatment. Data are presented in Table 5.
2) Results regarding daphnids introduced as adults (7 days old)
In the control 10 % immobility was observed. In addition, 30 % immobility was observed in the concentrations of 0.053 and 0.107 and 20 % immobility was observed in the highest test concentration of 0.470 mg/L. However, the control immobility of 10 % was compensated by Abbott`s formula, reducing the immobility to 20 % and 10 %, respectively. There was no significant difference in survival of individuals between the control and any treatment. Data are presented in Table 6.
- Reproductive output
1) Results regarding daphnids introduced as juveniles (<24h old)
There was no significant effect on the reproduction of surviving Daphnia magna over the range of the tested concentrations. The cumulative number of living offspring per introduced parent ranged from 91.8 in the control to 82.9 in the treatments. Data are presented in Table 7 and 8.
2) Results regarding daphnids introduced as adults (7 days old)
There was no significant effect on the reproduction of surviving Daphnia magna over the range of the tested concentrations. The cumulative number of offspring per introduced parent ranged from 95.7 to 126.9 over all treatments. Data are presented in Table 9 and 10.

- Age at first brood
1) Results regarding daphnids introduced as juveniles (<24h old)
Age at the first brood was between 8.9 days in the control and 9.6 days in the highest treatment. A significant effect on the age at the first reproduction between control and the highest treatment of 0.470 mg/L could be observed. Data are presented in Table 7 and 8.
2) Results regarding daphnids introduced as adults (7 days old)
No significant effect on the age at the first reproduction between control and any treatment could be observed. Age at the first brood was between 5.5 days and 5.9 days over all treatments. Data are presented in Table 9 and 10.

- Intrinsic rate of population increase
1) Results regarding daphnids introduced as juveniles (<24h old)
The intrinsic rate ranged from 0.322 to 0.339 over all treatments and was not statistically significant different from the control. Data are presented in Table 7 and 8.
2) Results regarding daphnids introduced as adults (7 days old)
The intrinsic rate ranged from 0.403 to 0.454 over all treatments and was not statistically significant different from the control. Data are presented in Table 9 and 10.

- Development rate
1) Results regarding daphnids introduced as juveniles (<24h old)
The development rate ranged from 0.105 to 0.113 over all treatment levels. The highest concentration of 0.47 mg/L was statistically significant different from the control. Data are presented in Table 7 and 8.
2) Results regarding daphnids introduced as adults (7 days old)
The development rate ranged from 0.171 to 0.182 over all treatment levels and was not statistically significant different from the control. Data are presented in Table 9 and 10.


- Mortality of control: No mortality.
- Other adverse effects control: No.
- Immobilisation of control: No
- Abnormal responses: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The substance was not stable during the test duration. Within seven days the substance was degraded and the measured concentration was between 28.0 % and 52.5 % of the nominal concentration. For the two lowest concentrations no test item could be quantified from day 14 on (LOQ = 0.003 mg Cyanamide/L). Details are presented in Table 1 and 2.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Toxic reference standard
In order to confirm the sensitivity of the test species Daphnia magna, acute immobilization tests over 24 h with the reference substance (RS) K2Cr2O7 are performed in regular intervals, as proposed by OECD 202. The results of the latest reference study (December 2018) are in agreement with historical 24 h EC50-values obtained in this institute, and the stated range given in the OECD guideline (EC50 = 0.6 – 2.1 mg/L).

Immobilization after 24 h:
Control: 0 %
0.40 mg RS/L: 10 %
0.60 mg RS/L: 20 %
0.90 mg RS/L: 20 %
1.35 mg RS/L: 45 %
2.00 mg RS/L: 100 %

24 h EC50 value:
Immobilization: 1.126 mg/L (95% CL : 0.653 – 1.942 mg/L)

Reported statistics and error estimates:

Evaluation and statistics
Numerical values are frequently rounded to a smaller degree of precision (number of digits) than used in the actual calculation. Minor differences in results obtained from calculations with such rounded values in comparison to those obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and thus of no practical concern.

Statistical calculations:
For each endpoint, the NOEC and LOEC. A LOEC was calculated by using ANOVA followed by Student-t test, Fisher`s Exact Binomial test, Dunnett’s or Williams’ test or an appropriate non-parametric test.
Since no statistically significant concentration-response relationships could be observed for any endpoint, no ECx analysis could be computed.
The computer software ToxRat® [9] was used for statistical evaluations.
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations was based on the initially measured concentrations.

The following endpoints observed in the reproduction test were evaluated quantitatively:

• Immobility of parental generation daphnids
• Time to the first brood
• Cumulative number of live offspring at day 21 (study end)
• Individual length of adults

At the end of the test, the total number of living offspring produced per parent daphnia was counted excluding the number of offspring in the analysis from parent daphnids which died accidentally and/or inadvertently during the test. According to OECD 211, it is required to report the NOEC/EC for the cumulative offspring per survived parent since no significant trend in mortality of adults.

Analytical results on measurements

Table 1:      Measured concentrations of Cyanamide during the 21-day exposure period (conc. 1 - 3).

Samp-ling time	Nominal [mg/L]	Meas-ured [mg/L]	of nominal [%]	Nominal [mg/L]	Meas-ured [mg/L]	of nominal [%]	Nominal [mg/L]	Meas-ured [mg/L]	of nominal [%]
0h	0.025	0.026	104	0.05	0.053	106	0.1	0.107	107
1h		0.026	104		0.053	106		0.103	103
2h		0.025	100		0.052	104		0.106	106
4h		0.026	104		0.053	106		0.119	119
24h		0.024	96.0		0.049	98.0		0.099	99.0
48h		0.022	88.0		0.041	82.0		0.091	91.0
4d		0.016	64.0		0.033	66.0		0.067	67.0
7d		0.007	28.0		0.019	38.0		0.041	41.0
14d		<LOQ*	-		<LOQ*	-		0.007	7.0
21d		<LOQ*	-		<LOQ*	-		<LOQ*	0.0

* LOQ = 0.003 mg Cyanamide/L

Table 2:      Measured concentrations of Cyanamide during the 21-day exposure period (conc. 4 – 5).

Sampling time	Nominal [mg/L]	Measured [mg/L]	of nominal [%]	Nominal [mg/L]	Measured [mg/L]	of nominal [%]
0h	0.2	0.218	109	0.4	0.470	118
1h		0.223	112		0.432	108
2h		0.219	110		0.470	118
4h		0.242	121		0.470	118
24h		0.208	104		0.441	110
48h		0.189	94.5		0.400	700
4d		0.144	72.0		0.317	79.3
7d		0.091	45.5		0.210	52.5
14d		0.015	7.5		0.046	11.5
21d		<LOQ*	-		0.017	4.3

* LOQ = 0.003 mg Cyanamide/L

 

 

Length

Table3:      Growth data and percent alteration of length compared to controls after 21 days for daphnids introduced as juveniles.

Cyanamide [mg/L]	Length on day 21 Mean ± SD [mm]	Decreasein length [%]
Control	4.36 ± 0.224	-
0.026	4.06 ± 0.112 (-)	6.9 (-)*
0.053	4.33 ± 0.220 (-)	0.6 (-)
0.107	4.24 ± 0.217 (-)	2.6 (-)
0.218	4.12 ± 0.207 (-)	5.4 (-)*
0.470	4.27 ± 0.256 (-)	2.1 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

* Statistical evaluations revealed a significant effect on length for this concentration. However, only low inhibitions of 6.9 % at the lowest concentration of 0.026 mg Cyanamide/L, and 5.4 % at a concentration of 0.218 mg Cyanamide/L were observed. However, only low inhibitions of 6.9 % at a concentration of 0.026 mg Cyanamide/L and 5.4 % at a concentration of 0.218 mg Cyanamide/L were observed. According to expert judgement, these values were considered not to be significantly different from the control since effects below 10 % compared to control are not considered to be ecotoxicologically relevant.

 

Table4:      Growth data and percent alteration of length compared to controls after 21 days for daphnids introduced as adults.

Cyanamide [mg/L]	Length on day 21 Mean ± SD [mm]	Decreasein length [%]
Control	4.51 ± 0.226	-
0.026	4.32 ± 0.221 (-)	4.3 (-)
0.053	4.14 ± 0.307 (-)	8.2 (-)*
0.107	4.22 ± 0.132 (-)	6.6 (-)*
0.218	4.37 ± 0.257 (-)	3.2 (-)
0.470	4.40 ± 0.298 (-)	2.5 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

 (+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

* Statistical evaluations revealed a significant effect on length for this concentration. However, only low inhibitions of 8.2 % at a concentration of 0.053 mg Cyanamide/L and 6.6 % at a concentration of 0.107 mg Cyanamide/L were observed. According to expert judgement, these values were considered not to be significantly different from the control since effects below 10 % compared to control are generally not considered to be ecotoxicologically relevant.

 

 

Immobilisation

Table5:      Survival data and percent reduction of survival compared to the control after 21 days for daphnids introduced as juveniles.

Cyanamide [mg/L]	Parental survival [number of individuals]	Reduction of survival [%] (immobility)
Control	10	0
0.026	8 (-)	20 (-)
0.053	9 (-)	10 (-)
0.107	9 (-)	10 (-)
0.218	9 (-)	10 (-)
0.470	10 (-)	0 (-)

 (+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments

 

Table6:      Survival data and percent reduction of survival compared to the control after 21 days for daphnids introduced as adults.

Cyanamide [mg/L]	Parental survival [number of individuals]	Reduction of survival [%] (immobility)	Reduction of survival [%] (immobility) compensated after Abbot`s formula
Control	9	10	0
0.026	10 (-)	0	0 (-)
0.053	7 (-)	30	20 (-)
0.107	7 (-)	30	20 (-)
0.218	10 (-)	0	0 (-)
0.470	8 (-)	20	10 (-)

 (+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments

 

Table7:      Reproduction data for daphnids introduced as juveniles.For raw data see Annex A1.

Cyanamide [mg/L]	Cumulative offspring per surviving parent Mean ± SD [Ind.]	Age at first brood Mean ± SD [days]	Development rate Mean ± SD [Ind.]	Intrinsic rate Mean ± SD
Control	91.8 ± 19.0	8.9 ± 0.70	0.113 ± 0.009	0.322 ± 0.026
0.026	82.9 ± 7.41 (-)	9.1 ± 0.70 (-)	0.111 ± 0.010 (-)	0.334 ± 0.012 (-)
0.053	88.7 ± 22.2 (-)	8.9 ± 0.73 (-)	0.113 ± 0.010 (-)	0.326 ± 0.042 (-)
0.107	88.3 ± 18.1 (-)	9.3 ± 0.92 (-)	0.108 ± 0.010 (-)	0.339 ± 0.019 (-)
0.218	88.2 ± 11.0 (-)	9.2 ± 0.50 (-)	0.109 ± 0.006 (-)	0.325 ± 0.032 (-)
0.470	89.3 ± 23.1 (-)	9.6 ± 0.88 (-)*	0.105 ± 0.009 ()**	0.333 ± 0.032 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

*Statistical evaluations revealed a significant effect on age of first reproduction for this concentration. However, only a low inhibition of 6.8 % at this concentration of 0.0470 mg Cyanamide/L was observed. According to expert judgement, this inhibition was considered not to be significantly different from the control since effects below 10 % compared to control are generally not considered to be ecotoxicologically relevant

** Statistical evaluations revealed a significant effect on development rate for this concentration. However, only a low inhibition of 7.2 % at this concentration of 0.047 mg Cyanamide/L was observed. According to expert judgement, this inhibition was considered not to be significantly different from the control since effects below 10 % compared to control are generally not considered to be ecotoxicologically relevant (according to OECD and EFSA [1], [2]).

Table8:     Percent reduction of reproduction compared to controls after 21 days for daphnids introduced as juveniles.

Cyanamide [mg/L]	Reduction of reproduction per surviving parent [%]	Minimum detectable difference to control in age of first reproduction [%]	Inhibition of development rate [%]	Inhibition of intrinsic rate [%]
Control	-	-	-	-
0.026	9.7 (-)	6.3 (-)	2.2 (-)	-3.9 (-)
0.053	3.4 (-)	6.8 (-)	0.5 (-)	-1.3 (-)
0.107	3.8 (-)	6.7 (-)	4.1 (-)	-5.2 (-)
0.218	3.9 (-)	6.9 (-)	3.2 (-)	-1.1 (-)
0.470	2.7 (-)	6.8 (-)*	7.2 (-)**	-3.5 (-)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments

*Statistical evaluations revealed a significant effect on age of first reproduction for this concentration. However, only a low inhibition of 6.8 % at this concentration of 0.0470 mg Cyanamide/L was observed. According to expert judgement, this inhibition was considered not to be significantly different from the control since effects below 10 % compared to control are generally not considered to be ecotoxicologically relevant.

** Statistical evaluations revealed a significant effect on development rate for this concentration. However, only a low inhibition of 7.2 % at this concentration of 0.0470 mg Cyanamide/L was observed. According to expert judgement, this inhibition was considered not to be significantly different from the control since effects below 10 % compared to control are generally not considered to be ecotoxicologically relevant.

 

Table9:      Reproduction data for daphnids introduced as adults.For raw data see Annex A1.

Cyanamide [mg/L]	Cumulative offspring per surviving parent Mean ± SD [Ind.]	Age at first brood Mean ± SD [days]	Development rate Mean ± SD [Ind.]	Intrinsic rate Mean ± SD
Control	108.0 ± 19.9	5.9 ± 0.88	0.171 ± 0.021	0.407 ± 0.060
0.026	109.9 ± 21.2 (-)	5.7 ± 0.63 (-)	0.177 ± 0.015 (-)	0.444 ± 0.045 (-)
0.053	108.6 ± 43.5 (-)	5.8 ± 0.71 (-)	0.176 ± 0.017 (-)	0.403 ± 0.076 (-)
0.107	95.7 ± 15.9 (-)	5.5 ± 0.00 (-)	0.182 ± 0.000 (-)	0.422 ± 0.061 (-)
0.218	114.8 ± 26.2 (-)	5.9 ± 0.84 (-)	0.172 ± 0.020 (-)	0.447 ± 0.066 (-)
0.470	126.9 ± 36.6 (-)	5.5 ± 0.00 (-)	0.182 ± 0.000 (-)	0.454 ± 0.074 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments; SD: standard deviation

 

 

Table10:   Percent reduction of reproduction compared to controls after 21 days for daphnids introduced as adults.

Cyanamide [mg/L]	Reduction of reproduction per surviving parent [%]	Inhibition of development rate [%]	Inhibition of intrinsic rate [%]
Control	-	-	-
0.026	-1.8 (-)	-3.5 (-)	-9.3 (-)
0.053	-0.5 (-)	-2.8 (-)	1.0 (-)
0.107	11.4 (-)	-6.3 (-)	-3.9 (-)
0.218	-6.3 (-)	-0.6 (-)	-10.0 (-)
0.470	-17.5 (-)	-6.3 (-)	-11.6 (-)

(+) statistically significant difference between controls / (-) no significant difference between controls and treatments

 

 

Derived effect concentrations

1)   Daphnids introduced as juveniles (<24h old)

For the assessment of effects and for calculation of effect concentrations the mean numbers of offspring per concentration were used. Since no statistically significant concentration-response relationships could be observed for any concentration, no ECx analysis could be computed for any endpoint.

Thus, the LC₁₀for immobilisation was determined to be > 0.470 mg/L. In addition, EC₁₀for reproduction, length, development rate and intrinsic rate were determined to be > 0.470 mg/L.

In addition, no statistically significant effects between treatments and control were observed for reproduction of surviving parent daphnids, immobilisation, length and intrinsic rate r, resulting in a NOEC of ≥ 0.470 mg/L and a LOEC of > 0.470 mg/L for each endpoint. The NOEC for age of first reproduction and development rate was calculated to be of 0.218 mg/L, respectively. However, due to a low inhibition of 6.8 % and 7.2%, the NOEC was set to be 0.47 mg/L since effects below 10 % compared to the control are not considered to be ecotoxicologically relevant.

The effective concentrations based on the initially measured Cyanamid concentrations are summarized inTable11.

 

Table11:   Effective concentrations based on initially measured Cyanamide concentration for the exposure ofDaphnia magnaintroduced as juveniles for 21 days.

Parameter		EC50	EC20	EC10	LOEC	NOEC
		[mg Cyanamide/L]
Offspring per survivng parent	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Immobility	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Length	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Age of first reproduction	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Development rate	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Intrinsic rate	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-

n.d.: not determined due to mathematical reasons or inappropriate data, for more information see Annex 2.

¹⁾The age of first reproduction does not allow to calculate ECx-values because it is not possible to define the effect size x (maximum possible increase in age not known). The variable was only evaluated by hypothesis testing to determine an LOEC/NOEC.

The NOEC for age of first reproduction and development rate was calculated to be 0.218 mg/L, respectively. However, due to low inhibition rates of 6.8% and 7.2% at a concentration of 0.218 mg/L, the NOEC was set to be higher than 0.47 mg/L by expert judgement, since effects below 10 % compared to control are generally not considered to be ecotoxicologically relevant and it is generally recommended by OECD and EFSA to use the EC10 approach in preference to the NOEC approach for the environmental risk assessment [1], [2].

 

2)   Daphnids introduced as adults (7 days old)

For the assessment of effects and for calculation of effect concentrations the mean numbers of offspring per concentration were used. Since no statistically significant concentration-response relationships could be observed for any concentration, no ECx analysis could be computed for any endpoint.

Thus, the LC₁₀for immobilisation was determined to be > 0.470 mg/L. In addition, EC₁₀for reproduction, length, development rate and intrinsic rate were determined to be > 0.470 mg/L.

In addition, no statistically significant effects between treatments and control were observed for any observed endpoint including reproduction of surviving parent daphnids, age at first reproduction, immobilisation, length, development rate and intrinsic rate r, resulting in a NOEC of ≥ 0.470 mg/L and a LOEC of > 0.470 mg/L for each endpoint.

 

The effective concentrations based on the initially measured Cyanamid concentrations are summarized inTable12.

 

Table12:   Effective concentrations based on initially measured Cyanamide concentration for the exposure of Daphnia magna introduced as juveniles for 21 days.

Parameter		EC50	EC20	EC10	LOEC	NOEC
		[mg Cyanamide/L]
Offspring per survivng parent	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Immobility	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Length	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Age of first reproduction ^1)	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Development rate	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-
Intrinsic rate	Value	n.d.	n.d.	n.d.	> 0.47	≥ 0.47
	95 %-cl lower	n.d.	n.d.	n.d.	-	-
	95 %-cl upper	n.d.	n.d.	n.d.	-	-

n.d.: not determined due to mathematical reasons or inappropriate data, for more information see Annex 2.

¹⁾The age of first reproduction does not allow to calculate ECx-values because it is not possible to define the effect size x (maximum possible increase in age not known). The variable was only evaluated by hypothesis testing to determine an LOEC/NOEC.

 

[1] OECD Guideline for Testing of Chemicals 201, Sect. 2: Effects on Biotic Systems, Freshwater Alga and Cyanobacteria, Growth Inhibition Test. 23 March 2006. Annex 5, corrected 28 July, 2011.

[2] EFSA (European Food Safety Authority) (2009). Scientific Opinion of the Panel on Plant Protection Products and their Residues on a request from EFSA updating the opinion related to Annex II and III: Ecotoxicological studies. The EFSA Journal,1165-1190. doi:10.2903/j.efsa.2009.1165.

Validity criteria fulfilled:

yes

Conclusions:

For the endpoints offspring per surviving parent, immobility and intrinsic rate of population increase (r) no effect was observed in the tested concentrations.
21-d NOEC ≥ 0.470 mg cyanamide/L
21-d LOEC > 0.470 mg cyanamide/L
Initial test concentrations (0h) of 0.026, 0.053, 0.107, 0.218 and 0.470 mg cyanamide/L (104 – 118% of nominal) were used for evaluation. During the 21-day exposure period, concentrations decreased to 0 – 4.3 % of nominal at test end.

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 detailed description where read across is used/recommended and where it is preferable to refrain 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:

Cyanamide is the first metabolite of the calcium cyanamide containing fertiliser product PERLKA. To refine the aquatic risk assessment for the agricultural application of PERLKA, a non-standard test was developed at the Fraunhofer Institute for Molecular Biology and Applied Ecology. The aim of this study was to investigate the influence of cyanamide on the reproduction of aquatic invertebrates, represented byDaphnia magna, under realistic exposure conditions as part of the refined risk assessment for cyanamide release from PERLKA. Therefore, the test item cyanamide - which is the main transformation product of calcium cyanamide - was applied once in a water sediment system under static test conditions. The test was performed based on OECD guideline 211 with two different life stages (juvenile daphnids aged less than 24 hours and adult daphnids with an age of approx. 7 - 8 days which started to reproduce). Young and adult female daphnids were exposed to the test item added to water at five concentrations for 21 days. Effects on reproductive performance, survival, growth (adult length at test termination) development rate and intrinsic rate of population increase were investigated.

For the investigations, the daphnids were exposed to a control and five nominal concentrations: control, 0.026, 0.053, 0.107, 0.218 and 0.470 mg cyanamide/L. The nominal test concentrations were prepared in Cu-reduced dilution water. To assess the actual test item concentrations sampling of test solutions were performed at test start and 1, 2, 4, 24 and 48 hours after starting the test and furthermore after 4, 7, 14 and 21 days (test end). The concentrations of the test item in the aqueous phase of all treatment levels were determined by LC-MS/MS (LOQ = 0.003 mg/L). Measured initial cyanamide concentrations were between 104 – 118 % of nominal. During the 21-day exposure period, concentrations decreased to 0 – 4.3 % of nominal at test end (see Table 1 and 2).

The ECx concentrations could not be calculated, as for the endpoints offspring per surviving parent, immobility and intrinsic rate of population increase no effect was observed in the tested concentrations. The 21-day NOECs based on offspring per surviving parent, immobility and intrinsic rate of population increase are therefore equal to or higher than the highest concentration tested 0.470 mg cyanamide/L (initial measured test item concentration). The sublethal effects investigated were: reproductive output, intrinsic rate of population increase, development rate, age at first brood and body length. The endpoint body length was excluded as the effects did not reflect a monotone dose-response.

The NOEC for development rate and age at first brood were presented in the report as being as well equal to or above the highest tested concentration based on expert judgement regarding the low effect (below 10 %). Considering the statistical evaluation, a significant effect was determined for the highest tested concentration, hence, a NOEC equalling 0.218 mg cyanamide/L and a LOEC of 0.470 mg cyanamide/L would be derived from statistical evaluation.

Production of offspring in the treated groups indicated that cyanamide did not have a significant effect on the reproduction at concentrations equal to or greater than 0.470 mg cyanamide/L (initial test concentration). 

The most sensitive endpoints were age at first brood and development rate. Considering that the difference of age at first brood causing a significant effect but the effect for reproductive output is not significantly deviating in the tested concentrations from the control, the endpoint “Age at first brood” is not regarded as key value for risk assessment. The endpoint development rate was not defined in the report and in the OECD 211 test guideline, the calculation is not given in the report. Hence, this value is not taken forward to risk assessment.

  

Results Synopsis

 

Test Organism Age (e.g. 1st instar): Test design 1):≤24 hours (juvenile); Test design 2): 7 to 8 days old (adult)

Test Type: Static water-sediment system

 

NOEC and LOEC based on initial concentrations according to report (results for juvenile and adult daphnids are the same, if not otherwise indicated):

21-d NOEC (offspring per surviving parent) ≥ 0.470 mg cyanamide/L initial test conc.

21-d LOEC (offspring per surviving parent) > 0.470 mg cyanamide/L initial test conc.

 

21-d NOEC (immobility) ≥ 0.470 mg cyanamide/L initial test conc.

21-d LOEC (immobility) > 0.470 mg cyanamide/L initial test conc.

 

21-d NOEC (Age of first reproduction) ≥ 0.470 mg cyanamide/L initial test conc.^*)

21-d LOEC (Age of first reproduction) > 0.470 mg cyanamide/L initial test conc.^*)

 

21-d NOEC (Development rate) ≥ 0.470 mg cyanamide/L initial test conc.^*)

21-d LOEC (Development rate) > 0.470 mg cyanamide/L initial test conc.^*)

 

21-d NOEC (Intrinsic rate r) ≥ 0.470 mg cyanamide/L initial test conc.

21-d LOEC (Intrinsic rate r) > 0.470 mg cyanamide/L initial test conc.

 

^*)NOEC and LOEC values for juvenile daphnids were set higher in the report by expert judgement, because the significant effect was causing an effect below 10 %. Keeping the statistical significance as criterium for NOEC and LOEC derivation the NOEC would equal for both 0.218 mg/L and the LOEC for both would equal 0.470 mg cyanamide/L initial test conc.

 

 

Endpoint(s) Effected: 

-Reproductive output (Def.: number of living offspring produced by parental animals within the test period)

-  Age at first brood

-  Intrinsic rate of population increase (Def.: measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations it will be zero. For growing populations it will be positive and for shrinking populations it will be negative. Clearly the latter is not sustainable and ultimately will lead to extinction.)

-  Body length (results excluded from derivation of effect values as no dose-response was shown)

-  Immobilisation

Description of key information

In the absence of chronic data on aquatic invertebrates for calcium cyanamide, information from the read-across substance cyanamide is used.

 

The long-term toxicity of cyanamide to aquatic invertebrates (Daphnia magna) was examined in two available studies. In the study from Murrell (1995) the growth and reproduction of D. magna were assessed in an unaerated, flow-through, 21-day test. An EC50 of > 0.41 mg cyanamide/L and NOEC of 0.1044 mg cyanamide/L were calculated from the obtained results.

 

In addition, a non-standard D. magna reproduction study (based on the OECD guideline 211) was conducted (Brüggemann, 2019). The study was developed as part of the refined risk assessment for cyanamide released from the calcium cyanamide fertiliser product PERLKA to investigate the effect of cyanamide on the reproduction of D. magna under more realistic conditions. For simulating a typical run-off-situation after application of PERLKA to agricultural fields, cyanamide was applied to an aerated water sediment system once under static test conditions. The test item was applied to two different life stages of D. magna (juvenile daphnids aged less than 24 hours and adult daphnids with an age of approx. 7 - 8 days which started to reproduce). Effects on reproductive performance, survival, growth (adult length at test termination), development rate, and intrinsic rate of population increase were investigated. No effect was seen on the reproduction neither for juvenile nor for adult daphnids exposed. The 21-d NOEC (offspring per surviving parent) equals or is above 0.470 mg cyanamide/L initial measured test concentration.

 

The more sensitive value reported by Murrell (1995) is forwarded as key value to the chemical safety assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.104 mg/L

Additional information

Upon dissolution in water calcium cyanamide is rapidly converted 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 detailed description where read across is used/recommended and where it is preferable to refrain 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"

 

No chronic toxicity values are available for calcium cyanamide. Therefore, available data (two studies similar to OECD guideline 211) for cyanamide are used for the chemical safety assessment:

Murrell 1995:

The effects of cyanamide on the growth and reproduction of Daphnia magna (< 24-hours old) were assessed in an unaerated, flow-through, 21-day test. Treatments consisted of a water control and nominal concentrations of 0.025, 0.05, 0.1, 0.2 and 0.4 mg cyanamide/L. Forty daphnids (4 replicates contain 10 daphnids) were exposed to each test level.

All chemical and physical parameters (dissolved oxygen concentration, temperature) in the definitive test were within expected ranges. Mean measured concentrations of cyanamide were 0.023, 0.049, 0.1, 0.21 and 0.41 mg /L and ranged from 92 to 105 % of nominal concentrations. In the test medium the test item was sufficiently stable during the test period of 21 days.

Survival of Daphnia magna was not significantly affected in any test level when compared to the control. The 21-d EC50 based on immobility was > 0.41 mg cyanamide/L (based on mean measured test concentrations).

The 0.023 and 0.41 mg/L test levels were significantly different when compared to the control for adult length. The length was not significantly different in the other concentrations compared to the control. The data for the 0.023 mg/L test level were considered aberrant because they did not follow a normal dose-response pattern. Also, the 0.023 mg/L test level had the second highest number of young/adult/reproduction day of all the test levels and control. The significant difference at the 0.41 mg/L test level is considered a test material effect.

The mean weights were not significantly different from the control at any test concentration. The time to first brood was 7 days for the control and all test concentrations. Thus, it can be concluded that the time to first brood is not influenced by cyanamide.

The 0.41 mg/L test level was significantly different when compared to the control for the reproduction mean. The remaining levels were not significantly different from the control. There were no sublethal (behavioural) effects noted at any test concentration.

Based on the effects on length and reproduction mean at the 0.41 mg/L test level, the EC50 can be identified as 0.41 mg/L (based on mean measured test concentrations). Based on the lack of weight, length, survival and reproduction effects, the >0.1 mg/L (0.1044 mg/L) test level can be identified as NOEC.

 

The exposure regime in the Murrell (1995) study does not reflect the environmental exposure in relation to the application frequency of the cyanamide containing biocidal product ALZOGUR (max. two applications per year) and the plant growth regulator DORMEX (single annual application) or the calcium cyanamide based fertilizer PERLKA (single annual application). Tehrefore, an additional study (Brüggemann 2019) was conducted to examine the chronic effects of cyanamide to the most sensitive aquatic organism, i.e. D. magna, under a more realistic exposure regime.

 

Brüggemann 2019:

Conducting the Daphnia magna reproduction test with cyanamide using a modified exposure regime in a water-sediment system is justified based on the legal principles of § 19, chapter 1 b) iv) of Regulation (EU) No 528/2012 in relation to the upcoming product authorisation procedures for the biocidal product ALZOGUR. For the plant growth regulator DORMEX AlzChem Trostberg GmbH holds registrations in various non-EU countries. Furthermore, this information is essential for refining the environmental risk assessment of DORMEX in the context of renewal of existing registrations and of obtaining new registrations (e.g. in China).

Cyanamide is the transformation product originating from calcium cyanamide upon contact with water. Therefore, ECHA has requested AlzChem to consider available data for the transformation product cyanamide in the (eco)toxicological assessment of calcium cyanamide. According to REACH § 12, chapter 1, a technical dossier shall include all physicochemical, toxicological and ecotoxicological information that is relevant and available to the registrant.

This test complements the results obtained from the mesocosm study described in section 6.6 “Aquatic mesocosm study_420-02-2_Cyanamide_2019 (Hommen)” and is considered in the PNEC derivation for cyanamide and calcium cyanamide.

To refine the aquatic risk assessment of cyanamide released from the fertiliser product PERLKA a non-standard test was developed at the Fraunhofer Institute for Molecular Biology and Applied Ecology. The aim of this study was to investigate the influence of cyanamide on the reproduction of aquatic invertebrates, represented by D. magna, under realistic conditions as part of the refined risk assessment for cyanamide release from PERLKA. The exposure took place in a water-sediment system which is considered to reflect the conditions of a worst-case runoff scenario in agricultural applications. The test was performed based on OECD guideline 211 with two different life stages (juvenile daphnids aged less than 24 hours and adult daphnids with an age of approx. 7 - 8 days which started to reproduce), to evaluate potential differences in the response depending on the age of exposed daphnia. Juvenile and adult individuals of D. magna were exposed to the test item added to water at five concentrations for 21 days. Effects on reproductive performance, survival, growth (adult length at test termination) development rate and intrinsic rate of population increase were investigated.

 

The test was performed based on OECD guideline 211, deviating in the following aspects:

- Two different life stages (juvenile daphnids aged less than 24 hours and adult daphnids with an age of approx. 7 - 8 days which started to reproduce) were tested

- 10 pseudoreplicates and 2 true replicates;

- Sediment was introduced to the test system;

- Static system (no change of medium during test period);

- Instead of time-weighted mean concentrations, the starting concentration was used as basis for NOEC calculations in the report.

For the investigations, daphnids were exposed to a control and five nominal concentrations: control, 0.026, 0.053, 0.107, 0.218 and 0.470 mg cyanamide/L. The nominal test concentrations were prepared in Cu-reduced dilution water. To assess the actual test item concentrations sampling of test solutions were performed at test start and 1, 2, 4, 24 and 48 hours after starting the test and furthermore after 4, 7, 14 and 21 days (test end). The concentrations of the test item in the aqueous phase of all treatment levels were determined by LC-MS/MS (LOQ = 0.003 mg/L). Measured initial cyanamide concentrations were between 104 – 118 % of nominal. During the 21-day exposure period, concentrations decreased to 0 – 4.3 % of nominal concentrations at test end. During the 21-day exposure period, concentrations decreased to 0 – 4.3 % of nominal at test end.

The ECx concentrations could not be calculated, as for the endpoints offspring per surviving parent, immobility and intrinsic rate of population increase (r) no effect was observed in the tested concentrations. The 21-day NOECs based on offspring per surviving parent, immobility and intrinsic rate of population increase (r)are therefore equal to or higher than the highest concentration tested 0.470 mg cyanamide/L (initial measured test item concentration). The sublethal effects included were: reproductive output (Def.: number of living offspring produced by parental animals within the test period), intrinsic rate of population increase, development rate, age at first brood and body length. The endpoint body length was excluded as the effects did not reflect a monotone dose-response.

The NOEC for development rate and age at first brood were presented in the report as being as well equal to or above the highest tested concentration based on expert judgement regarding the low effect (below 10 %). The age at first brood in the control was 8.9 ± 0.70 days [mean ± standard deviation], and in the highest concentration 9.6 ± 0.88 days [mean ± standard deviation]. The prolonged age at first brood is not resulting in a significantly reduced reproductive output (i.e. offspring per surviving parent). Therefore, this endpoint is not considered as key endpoint. The assessed endpoint development” rate is not defined in the report and in the OECD 211 test guideline, the calculation is not given in the report. Hence, this value is not taken forward to risk assessment.