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Diss Factsheets

Administrative data

additional ecotoxicological information
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-01 to 2018-09-07
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
The aim of this study is to investigate effects of cyanamide on freshwater ecosystems by monitoring zooplankton, macroinvertebrates, phytoplankton, periphyton and macrophytes in lentic outdoor mesocosms (enclosure systems).

The mesocosms contained an indigenous assemblage of invertebrates (zooplankton, macroinvertebrates) and plants (phytoplankton, periphyton and macrophytes). In addition, three macrophyte species (Lemna sp., Myriophyllum spicatum and Mentha aquatica) were introduced in pots or swimming enclosures to monitor effects on growth. The test item was applied once. The concentration of the cyanamide was regularly monitored in the water.

Fifteen mesocosms located in a pond at the test site Mesocosm GmbH, were used for the study. Five of the mesocosms served as untreated controls while five levels of the test item (in two replicates) were treated with the test item. Fate and effects were monitored over twelve weeks (86 days) after the application.

The study was conducted in accordance with the OECD Guidance Document “Freshwater Lentic Field Tests” (OECD 2006) as well as the recommendations from the EFSA Aquatic Guidance Document (EFSA PPR 2013) and the Biocide guidance.

Observed effects of the test item are classified according to the EFSA PPR (2013) and ECHA Guidance (ECHA 2017) to allow the derivation of an ETO-PNEC Ecological Threshold Option Predicted No Effect Concentration) and an ERO-PNEC (PNEC based on the Ecological Recovery Option).
GLP compliance:
yes (incl. QA statement)
Type of study / information:
experimental aquatic mesocosm study

Test material

Specific details on test material used for the study:
- Chemical name: Cyanamide
- CAS number: 420-04-2

Results and discussion

Any other information on results incl. tables


Weather conditions and water temperature

Air temperature reached a maximum of monthly means during the in-life phase with 20.5 °C in July 2018 and a minimum temperature of 14.7 °C in September 2018. The sunshine was measured in minutes per day and summed up to the month sunshine-minutes. During the inlife phase June had the fewest sunshine minutes with 13002 and July the most with 19464 minutes. The minimum monthly sum of precipitation during the in-life phase was measured in June 2018 with 30.2 mm and the maximum monthly sum of 56.4 mm was measured in July The water level remained within the range of 110 cm ± 20 %, and thus, no additional water was added to mesocosms. The water temperature measured 50 cm below water surface reached a maximum during the study with 23.0 °C in the beginning of August and a minimum temperature of 15.1 °C in the beginning of September 2018. The weather on application day (June 13, 2018), during the application and the sampling procedures was as follows: no precipitation, complete cloud cover, air temperature 13 – 14 °C.


Water and sediment characterization

Prior to the initiation of this study a representative aliquot of pond water and a sub-sample of the mixed sediment were sampled and analysed for heavy metals (Mn, Pb, Cd, Cr, Ni, Hg, Zn), PCBs (6 components), pesticides (27 components) and selected chemical parameters (Na, K, S). The sediment was additionally analysed for particle size distribution and total organic carbon. No findings above the corresponding LOQ were detected for PCBs and pesticides in water or sediment. The TOC of the sediment was 2.1 %.


Water quality parameters during the study

The chemical parameters were measured from depth integrated water samples. The values for nitrate (MDL 0.4 mg/L) were in general below the method detection limit (MDL) except for day -2 when concentrations up to 1.6 mg/L were measured. For ammonia the values were in the range from below MDL (MDL 0.01 mg/L) up to 0.06 mg/L with a mean value of 0.03 mg/L. For phosphate the values ranged from below MDL (MDL 0.2 mg/L) up to 0.3 mg/L with a mean value of 0.21 mg/L. For hardness the mean value during the study was 2.44 °dH with a range of 1.2 to 4.0 °dH.


Fate and exposure

The quantitative measurements of cyanamide in mesocosm water were performed by liquid chromatography (LC) coupled to a quadrupole tandem mass spectrometer (LC-MS/MS). The validation of the analytical methods for the quantification of cyanamide in water was performed in compliance with GLP according to the European Commission guidance document SANCO/3029/99 rev. 4 (EC 2000). The limit of quantitation (LOQ) was determined to be 0.003 mg cyanamide/L. The limit of detection (LOD) was defined to be the lowest calibration point 0.001 mg cyanamide/L.


The recovery of the nominal concentrations of cyanamide in the enclosure water in the first three samplings were 80 %, 90 % and 97 % per average at 1, 3 hours and 1 day, respectively, after the application. The recovery rates increased with the nominal concentration. Since most of the single values were above 80 % of nominal and the mean recoveries were close to 100 %, the intended dosing is considered to be confirmed by the measurements and the nominal concentrations are used to describe the treatment levels. The variability between the replicates per test concentration was very low and the general pattern over time was similar over the different test concentrations. However, DT50 calculated by log-linear regression for the available values from day 1 to day 83 ranged between 3.3 and 27.5 d and showed a clear trend to increase with nominal concentration. An overall mean DT50 was estimated to be 13.1 days.





Thirty seven taxa (including stages) were determined in the zooplankton samples. Cyclopodid adults and copepodids could not be determined further. Also nauplius larvae of copepods could not be differentiated and were used as a separate taxon. For the phantom midge Chaoborus sp., larvae and pupae were differentiated but considered together for the evaluation. Also, the sum of copepodids and adults was evaluated. Nauplii were considered separately since, there might have been also nauplii of Diaptomidae. In the zooplankton 60 % of the organisms found were copepods, mainly nauplii. Also very abundant was the rotifer Keratella quadrata (24 % of total zooplankton abundance over all samples). Cladocera were less abundant. The eleven taxa built only approximately 3% of the total abundance. One species, the Cladocera species Alona sp., showed temporary and slight deviations from the controls already at a concentration of 0.032 mg/L. In 0.1 mg/L also Cyclopidae and in 0.32 mg/L a few other crustacean and rotifer species might have been slightly directly or indirectly affected. For two species, Alona sp. and a not determined rotifer, significant deviations from the control were found over at least two consecutive samplings and thus, were considered pronounced. The application of 1 mg/L had pronounced but temporary effects on several taxa and in the enclosures treated with 3.2 mg/L, long-term effects were found. The community level analysis revealed no effects of 0.032 mg/L, pronounced effects of 0.1 – 1.0 mg/L and long-term effects of 3.2 mg/L. However, the class 3A for the zooplankton community due to significance of the PCA sample scores on day 30 and 44 is related to slight, temporary effects on different taxa on different days, i.e. Cyclopidae only on day 30 and on Alona sp. on Day 44.



Thirty three taxa respectively stages from seven classes were differentiated in the samples of the macroinvertebrates. 40 % of the counted organisms were Chaoborus larvae (Insecta). Also abundant were Tubificidae (worm, Clitellata), Asellus aquaticus (water louse, Crustacea), Cloeon dipterum (mayfly, Insecta), Zygoptera (damselflies, Insecta), Lymnaeidae and Planorbidae (snails, Gastropoda). If stages could be clearly combined to one taxon (e.g. Chaoborus larvae and pupae), they were considered together for evaluation.


Chaoborus sp. was the most sensitive macroinvertebrate species with slight effects at 0.1 mg/L and pronounced but temporary effects up to 1 mg/L. No recovery until the end of the study was observed in 3.2 mg/L. Asellus aquaticus was less sensitive but more vulnerable than Chaoborus since it was not affected up to 0.32 mg/L but in contrast to Chaoborus no recovery within eight weeks was observed already in 1 mg/L. Other insecta, i.e. Chironomidae, Zygoptera and Cloeon dipterum were less sensitive with no or only slight effects at 1 mg/L. Snails and worms were not affected, except a potential slight temporary promotion of Tubificidae. The macroinvertebrate community structure was slightly affected at 0.1 and 0.32 mg/L, but long-term deviations from the controls were observed at 1 and 3.2 mg/L.



One hundred taxa were identified in the phytoplankton samples, covering bacteria (blue-green algae, Cyanophyta), Chromista (Bacillariophyceae, Dinophyceae, Chrysophyceae, Cryptophyceae, and Xanthophyceae), Plantae (green algae, Chlorophyceae, and Zygnematophyceae) and Protozoa (Euglenoidea, formerly Euglenophyceae). The dominating species were the Cryptophyceae Chroomonas acuta / Rhodomonas sp. and Cryptopmonas erosa + ovata, and the Chrysophyceae Chromulina minima, small Chrysophyceae (1 – 5 μm) and Desmarella moniliformis. About two thirds of the cells counted belong to these five taxa.

The application had mainly indirect effects on the phytoplankton, only Cryptophyceae showed clear direct effects which were slight at 0.032 and 0.1 mg/L, pronounced but  temporary at 0.32 and1.0 mg/L and long-term at 3.2 mg/L. Other algae groups and also the total phytoplankton were promoted, at 3.2 mg/L often long-term until the end of the study. In lower test concentration effects were usually temporary, only for one species, the green alga Tetraedron caudatum, also 1 mg/L had long-term promoting effects. However, the species was rare and reached also in 1 and 3.2 mg/L no relevant abundances in relation to total phytoplankton abundance. Due to the direct and indirect effects on many species at different times after application, the ordination indicated long-term effects on the community level in 0.32 mg/L and higher. Up to 0.1 mg/L no effects or only slight effects on a few species were found. The chlorophyll measurements support the results from the phytoplankton identification and counting.



The periphyton growing on glass slides was analysed only by chlorophyll a measurements. Significantly higher values were found only in 3.2 mg/L on days 12, 26 and 54 for total chlorophyll a, green algae ((Chlorophyceae, Euglenophyceae, Conjugatophyceae) and chromophytes (Bacillariophyceae, Chrysophyceae, Dinophyceae, Xanthophyceae). Since there was no recovery within eight weeks but within the study, the effect of 3.2 mg/L was considered class 5A+. Bluegreen chlorophyll a was only significantly higher on day 12 but the chlorophyll a concentration was also higher on day 26, which was considered effect class 3A+.


Chlorophyll a concentrations of Cryptophytes in the periphyton were low and the MDDs were usually too high to detect a reduction. Only on the last sampling date, reduced chlorophyll a values in 1.0 and 3.2 mg/L were significant. However, due to the very low values and the absence of a concentration related pattern, these data were not considered for effect classification.



Effects on macrophytes were assessed by monitoring the coverage of plant species

(considering also mats of filamentous algae) and by measuring frond numbers of Lemna in

small ring enclosures or shoot lengths of Myriophyllum and Mentha planted in pots. The MDDs of the macrophytes data were generally small and thus, allowed the assessment of effects for five species according Brock et al. (2015). However, except of a NOEC of 1 mg/L for total and Myriophyllum coverage on day 85 and for Mentha on day 7, no indications of reduced growth were found. The lower coverage of Myriophyllum at the end of the study is not considered to be caused by the treatment due the very similar growth in all test concentrations before and the very low MDD resulting in a significance of a not relevant difference. The single NOEC for Mentha is also not considered relevant. On a few occasions, mean values at 3.2 mg/L were higher than in the control, which might indicate a promoting effects. However, expect for the short-term promotion of Lemna, all significant differences were very small and only detected due to the unusually low MDDs.


Thus, except for Lemna, effects on macrophytes are considered unlikely (effect class 1). The temporary promotion of Lemna at 3.2 mg/L was considered a class 3A+ effect.


Indicators for community metabolism

Oxygen, pH and conductivity are affected by photosynthesis and respiration, respectively uptake of nutrients. Thus, lower oxygen concentrations and pH values and higher conductivity indicates reduced primary production. However, no effects on these indicator value were found.



Based on the biological results so called regulatory acceptable concentrations (RACs) were determined. From the effect classification (see attachment), the following ecological threshold option (ETO) and an ecological recovery option (ERO)-RAC was derived:

ETO-RAC = 0.1 mg/L

ERO-RAC = 0.32 mg/L 

The ERO-RAC of 0.32 mg/L is considered to be the threshold concentration at which no population showed long-term effects.

Applicant's summary and conclusion

In a GLP outdoor mesocosm study the ETO-RAC and ERO-RAC of Cyanamide was determined to be 0.1 mg/L and 0.32 mg/L respectively.
Executive summary:

A GLP outdoor mesocosm study was performed with the aim to investigate the effects of cyanamide, applied as Cyanamid F1000, on zooplankton, macroinvertebrates, phytoplankton, periphyton and macrophytes. In addition to the biological aspects, the fate of cyanamide was monitored in water. 


Fifteen enclosures located in a pond at the test site, were used for the study. The pond was prepared in October 2016 to allow the establishment of a diverse community. The stainless steel enclosures were pressed on May 11, 2018, 33 days before the test item application, into the sediment and contained about 1760 L water with a depth of 110 cm ± 20 %. Five of the enclosures served as untreated controls while five levels of the test item (in two replicates) were treated with the test item (0.032; 0.1; 0.32; 1.0; und 3.2 mg cyanamide/L).


The single application was conducted on June 13, 2018. Fate and effects were monitored over approximately twelve weeks after application. Chemical analysis of cyanamide was performed in the water phase. Zooplankton (in water samples) and macroinvertebrates (sampled using different sampling methods) were differentiated to species level if practical. Phytoplankton and periphyton were monitored by delayed fluorescence spectroscopy for chlorophyll a concentrations. Phytoplankton was also identified to species level when possible and counted using an inverted microscope. Macrophytes´ growth was determined by estimations of % area coverage. In addition, three species (Lemna sp., Myriophyllum spicatum and Mentha aquatica) were tested using in-situ bioassays. Potential effects of the test item were statistically analysed by the multiple t-test of Williams for differences in taxon abundances to controls and to derive no-observed-effect concentrations (NOECs). Minimum detectable differences (MDDs) were used to identify those taxa which allowed a reliable evaluation according to the Aquatic Guidance Document (EFSA PPR 2013). Diversity indices and ordination analysis (i.e. Principal Response Curves) were applied to analyse effects on the community level for zooplankton, macroinvertebrates, and phytoplankton. Based on the statistical findings and additional evaluation of the data, e.g. with respect to the presence of concentration response relations, the timing and duration of effects and the population dynamics, effects were classified according to the scheme proposed by EFSA PPR (2013) and Brock et al. (2015) in order to allow the derivation of Regulatory Acceptable Concentrations under the Ecological Threshold and Ecological Recovery Option (ETO-RAC, ERO-RAC, EFSA 2013).


One day after application, the recovery of the nominal concentrations of cyanamide in the enclosure water was 97 % per average and ranged between about 84 and 110 %. Thus, the intended initial exposure was confirmed and nominal test concentrations were used to characterize the exposure. The variability of the measured cyanamide concentrations between the replicates per test concentration was low and the general pattern of dissipation was similar over the different test concentrations. However, DT50 values varied between 3 and 28 days and increased with nominal concentration, providing an overall mean of 13 days.


As a result a large variety of species covering many relevant taxonomic groups of freshwater ecosystems were present in the test systems, representing zooplankton, macroinvertebrates (incl. crustaceans, insects, snails and oligochaetes), algae and macrophytes. For eight macroinvertebrate, nine zooplankton, nine phytoplankton and five macrophyte taxa, the calculated Minimum Detectable Differences (MDDs) were sufficiently low to allow a reliable analysis according to the proposal of Brock et al. (2015). Thus, the requirements of the EFSA aquatic guidance document (EFSA 2013) that at least eight potentially sensitive populations should allow a statistical evaluation of direct effects are clearly fulfilled. Direct effects of Cyanamide were found on crustaceans, insects and algae of the class Cryptophyceae. Indirect effects, i.e. increased abundances compared to controls, were observed for rotifers, Tubificidae and several classes of the phytoplankton, periphyton and Lemna sp.


At the level of 0.032 mg Cynamide/L almost all taxa and endpoints were not affected. Only the Cladocera Alona sp. of the zooplankton and the Cryptophyceae Chroomonas acuta / Rhodomonas the phytoplankton might have been slightly and temporarily affected (effect class 2). However, abundance of Alona was generally low and the NOECs < 0.032 mg/L were found only on the dates when the species was not absent in at least one control sample. No clear decline in abundance of Alona sp. was observed up to 0.32 mg/L. The reduction of Chroomonas acuta in 0.032 mg/L was restricted to day 6 and the mean was still within the range of the control.


At 0.1 mg Cynamide/L slight effects were found for Cyclopidae, Chaoborus larvae, and Chlorophyceae in addition to the effects found in 0.032 mg/L. Effects on the community structure of the zooplankton were considered pronounced but temporary (class 3A) since significant deviations were found on two consecutive samplings (day 30 and 44). However, no pronounced effects on populations were found. Effects on the macroinvertebrate community structure were slight (due to the slight effects on Chaoborus).


At 0.32 mg Cyanamide/L additional taxa showed slight effects and pronounced temporary direct or indirect effects (class 3A) were found for more taxa than in 0.1 mg/L, e.g. Alona sp., Chaoborus sp. And Chroomonas acuta, Chlorophyceae). Only the PRCs for the phytoplankton indicated a long-term effect on the community structure (class 5B).


At 1.0 mg/L some zooplankton taxa showed pronounced effects, some of these only at the end of the study. In such cases, the duration of these effects could not be assessed. From the macroinvertebrates, the damselflies (Zygoptera) might have been slightly affected and Asellus aquaticus was affected over more than eight weeks but recovered until the end of the study (class 5A). Effects on the macroinvertebrate community structure were considered long-term without full recovery (class 5B).


Finally, at 3.2 mg/L several taxa showed long-term effects, often until the end of the study. Crustaceans, insects and Cryptophyceae were directly affected while rotifers and the total phytoplankton were promoted until the end of the study. A temporary promotion was also found for Lemna sp. Other macrophytes as well as snails were not affected.


From the effect classification, the following effect concentrations are proposed to derive an ETO and an ERO-RAC: At 0.1 mg/L, no pronounced effects (class 3A or higher) on populations were found, only the effect on the zooplankton community structure was considered class 3A due to significance on two sampling dates. Thus, this concentration would still allow an estimation of a protective ETO-RAC for all populations tested. At 0.32 mg/L, pronounced effects on a few populations were found, but these were only temporary (class 3A). However, indicated by the ordination analysis, the phytoplankton community structure was affected until the end of the study, which might have been a result of the high dynamic of the phytoplankton and promotion of some species, especially green algae at the end of the study which were still rare compared to the total phytoplankton. Thus, the ecological relevance of these promotions of a few, not dominant algae species is probably small and the ERO-RAC could thus be derived also from this concentration considering that no population showed long-term effects.