Calcium cyanamide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-17 to 2010-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

Regulation (EC) No 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

adopted 17 July 1992

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Activated sludge was collected from a sewage treatment works A-2500 Baden, which has a waste-water catchment that is predominantly domestic. On arrival in the laboratory and after sedimentation the clear supernatant was decanted and the sludge was filled up with mineral medium to its original volume to reduce the carbon content. The sample was aerated by means of filtered compressed air before being used for the study. The suspended solids concentration was determined by filtering a 5 mL sample through a pre-dried and pre-weighed glass filter (Whatman GF/C). The filter with solids were dried at 105°C and re-weighed and the sludge solids determined by difference. The inoculum was not acclimatised or adapted to "CALCIUM CYANAMIDE" before exposure to the test substance in this study.

Duration of test (contact time):

ca. 28 d

Initial conc.:

95.9 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparation of the test substance
A first main experiment was conducted prior to the reported one. Addition of the test substance to the mineral medium at the start of the study changed the pH in the vessels containing test substance to 10.1. Measurements of CO2 evolution of the test vessels A3 and A4 revealed that no biodegradation of the test substance occurred. The toxicity control containing test substance and Na Benzoate was indicative of an inhibition. It was deemed that the basic pH of the test solutions impaired or even mortified the bacteria in the activated sludge. Therefore it was decided in agreement with the sponsor to repeat the experiment using the following method of preparation of the test substance:
The test substance media were prepared using the withdrawn mineral medium prepared on the day before (see 2.4). For each test suspension preparation (A3, A4, A3/4, TK6) 600 mL mineral medium were divided into two parts. One part was used to add the respective amount (nominal 287.7 mg) of test substance. The pH of these dispersions was adjusted with 32 % HCL (Merck) to 7.3-7.4. The second part was used for quantitative transfer of the pH adjusted suspension to the respective test vessel to obtain the final volume of 3 000 mL of inoculated mixture per vessel.
The nominal concentration of 287.7 mg test substance per three litre medium corresponds to 10 mg carbon per litre. Based on the Certificate of Analysis provided by the sponsor the organic carbon content of the test substance is 10.43 % per weight. The actually weighed mass of the test substance to the four vessels was 287.9 (A3), 287.6 (A4), 287.3 (A3/4pH) and 287.4 mg (TK6) per 3 L medium. This organic carbon concentration corresponds to a theoretical CO2 (ThCO2) yield of 36.7 mg/L.

Procedure
Pre-incubation: On day –1, the test vessels were filled with mineral medium containing activated sludge to a total volume of 3000 mL (NK1, NK2, PK5) and 2400 mL respectively (A3, A4, A3/4, TK6), to give a concentration of suspended solids of approximately 20 mg/L in the final mixture on Day 0.
The vessels were sealed and aerated overnight with CO2-free air to purge the medium of CO2 prior to the beginning of the study. Apparatus for carbon dioxide scrubbing and a mixture of CO2-free oxygen and CO2-free nitrogen, from gas cylinders (Linde Gase, A-2492 Eggendorf), in the correct proportions (20 % O2: 80 % N2) were used. The air flow was regulated for each vessel individually. Air flow was determined volumetrically in intervals. Adjustments were made as necessary to maintain a flow rate in the range of 30 to 100 mL per minute (1 – 3 bubbles per sec). As an added precaution, a 0.0125 M barium hydroxide solution was used as CO2 absorber. On Day 0, the relevant vessels were opened to add the test or reference substance. The pH adjusted test dispersions (600 mL each) were added to vessels A3, A4, A3/4 and TK6, the reference substance was added to vessels PK5 and TK6. The test solutions were continuously stirred during the test. The vessels were incubated in darkness until the end of the study and the medium continually supplied with CO2-free air. Barium hydroxide (Merck, D-64271 Darmstadt, Germany) was used to trap the CO2 formed by the degradation of the test substance. Three absorption bottles each containing 100 mL of 0.0125 M barium hydroxide solution, were connected in series to each 5-litre flask. On the days of CO2 measurement, the barium hydroxide absorbers closest to the test vessels were disconnected. The remaining absorbers were moved up one position and a new absorber containing fresh barium hydroxide solution was placed at the far end of the series. In case a substantial precipitation was seen in the first trap, also the second bottle was analysed.
Test duration: 28 days exposure, with measurement of the last sample on Day 29

Reference substance:

benzoic acid, sodium salt

Preliminary study:

A first main experiment was conducted prior to the reported one. Addition of the test substance to the mineral medium at the start of the study changed the pH in the vessels containing test substance to 10.1. Measurements of CO2 evolution of the test vessels A3 and A4 revealed that no biodegradation of the test substance occurred. The toxicity control containing test substance and Na Benzoate was indicative of an inhibition. It was deemed that the basic pH of the test solutions impaired or even mortified the bacteria in the activated sludge.

Test performance:

Validity of the test
The test is considered to be valid if the difference of extremes of replicate values of the removal of the test substance at the plateau, at the end of the test or at the 10-d window, as appropriate, is less than 20 % and if the percentage degradation of the reference substance has reached the pass level by day 14. These criteria were fulfilled. The guidelines require that the final cumulative CO2 yield from the blanks should not exceed 40 mg CO2/L and values greater than 70 mg CO2/L should trigger the critical checking of the experimental techniques. As the critical value of 70 mg CO2/L was by far not exceeded this event did not adversely affect the outcome of the study. Total mean blank CO2 production in this study was 50.8 mg CO2/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: pH adjusted to 7.3-7.4

Details on results:

Calcium cyanamide was not degraded within 28 days.

Results with reference substance:

The plateau of biodegradation was reached approximately by about day 10 and the degradation of the positive reference substance sodium benzoate exceeded the pass level of 60 % by day 5. The theoretical CO2 production of the positive control reached 77.4 % on day 14.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

According to the results of this study, Calcium cyanamide, technical grade (Kalkstickstoff) cannot be classified as readily biodegradable.

Executive summary:

The biodegradability of calcium cyanamide, technical grade (Kalkstickstoff) was analysed by the determination of carbon dioxide evolution at frequent intervals over a 28 day period. The method used was that described in part C.4-C of Regulation (EC) 440/2008 and in OECD guideline 301B. The pH was adjusted to pH 7.3–7.4 to allow a proper growth of microorganisms. The pH without adjustment was 10.1.

Results:

- The reference substance sodium benzoate was degraded by 77.4 % within 14 days.

- Degradation in the toxicity control, containing sodium benzoate and the test substance, was not indicative of inhibition of the microbial activity by the test substance.

- Calcium cyanamide was not degraded after 28 days of incubation at a temperature between 21.0 and 23.1 °C.

According to the results of this study, calcium cyanamide, technical grade (Kalkstickstoff) cannot be classified as readily biodegradable.

Description of key information

According to the results of a ready biodegradation test (CO₂ evolution), calcium cyanamide, technical grade (Kalkstickstoff) cannot be classified as readily biodegradable.

In addition, information from the structural analogue cyanamide is taken into account.
The result of the study of van der Hoeck (1988) showed that cyanamide can be regarded as not readily biodegradable by the terms of this test. A following study (Malta, 1990) showed that cyanamide was completely degraded within two weeks when it served as nitrogen source for degradation of a carbon-containing compound (sodium acetate), whereas the cyanamide degradation was rather slow when cyanamide served as both carbon and nitrogen source.

It can be concluded that the biodegradation of cyanamide and calcium cyanamide in a standard ready biodegradability test is prevented by the presence of another easily available nitrogen source. But, as rapid degradation of cyanamide could clearly be demonstrated under environmentally realistic conditions in two aerobic water/sediment model systems (see IUCLID section 5.2.2) and in soil (see IUCLID section 5.2.3), cyanamide can thus be considered as rapidly degradable according to the CLP Regulation (EC) No 1272/2008, Annex I sections 4.1.2.9.2 and 4.1.2.9.3. Therefore, cyanamide (and calcium cyanamide in a read-across approach) were concluded to be "readily biodegradable".

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"

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The biodegradability of calcium cyanamide, technical grade (Kalkstickstoff) was analysed by the determination of carbon dioxide evolution at frequent intervals over a 28 day period. The method used was that described in Part C.4-C of the Regulation (EC) 440/2008 and in the OECD guideline 301B. The pH was adjusted to pH 7.3 –7.4 to allow a proper growth of microorganisms. The pH without adjustment was 10.1.

Results:

- The reference substance sodium benzoate was degraded by 77.4 % within 14 days.

- Degradation in the toxicity control, containing sodium benzoate and the test substance, was not indicative of inhibition of the microbial activity by the test substance.

- Calcium cyanamide was not degraded after 28 days of incubation at a temperature between 21.0 and 23.1 °C.

According to the results of this study, calcium cyanamide, technical grade (Kalkstickstoff) cannot be classified as readily biodegradable.

Upon dissolution in water calcium cyanamide is fast transformed to hydrogen cyanamide. Therefore, the fate in natural aquatic environments can be expressed in terms of cyanamide, irrespective of the substance constituting the exposure source. Thus, read-across from cyanamide to calcium cyanamide is additionally performed:

Two ready biodegradable studies were conducted in which the newer study (Malta, 1990) examined the influence of some limiting factors on the biodegradability of cyanamide that might explain the not readily biodegradable result obtained by the former study (van der Hoeck, 1988).

 

In the study of van der Hoeck (1988) a stock solution of aqueous cyanamide solution was prepared in nutrient medium inoculated with activated sludge in two concentrations corresponding to 14.7 and 29.4 mg ai cyanamide/L. Control flasks without test substance and a sterile control (test medium autoclaved for 20 minutes at 120 °C) were included in the test. Sodium acetate was used as a positive control. The test samples were incubated for 28 days at 20°± 2 °C. The residual dissolved organic carbon (DOC) was measured using a TOC-analyser after persulphate oxidation to assess the percent of biodegradation. The biodegradability of sodium acetate was used as a measure for the microbial activity of the inoculum, and to detect any inhibition of this activity due to the presence of high concentrations of cyanamide.

Sodium acetate was completely degraded within one week i.e. biodegradation reached a level of 97 to 100 % indicating that sodium acetate is readily biodegradable by the terms of this test. The sodium acetate degradation was not influenced by cyanamide present in the medium. Cyanamide was not degraded in the inoculated medium within 28 days. Therefore, cyanamide is regarded as not readily biodegradable by the terms of this test. No degradation was found in the sterile control confirming the stability of the test substance. As literature data indicate that cyanamide should be readily biodegradable (in contrast to the results of this study), the influence of some limiting factors (i.e. nitrogen source, test substance and inoculum concentrations) on the biodegradability of cyanamide was determined in another study (Malta, 1990).

For this purpose in the study of Malta (1990), one nitrogen free medium was prepared leaving out NH4Cl and yeast extract for the testing of cyanamide as nitrogen source. Activated sludge was added to the medium and the test samples were incubated for 56 days at 20 ± 2°C. Evolving CO₂ was trapped in NaOH traps and its amount determined by titration with 0.1 M HCL.

Results showed that in the nitrogen limitation samples the cyanamide was completely degraded within two weeks when it served as a nitrogen source for degradation of a carbon-containing compound (sodium acetate). When cyanamide served as both carbon and nitrogen source it was degraded slowly with about 22 % within 8 weeks. Results of the samples that examined the influence of the test substance and the inoculum concentrations showed that neither the test substance concentration nor the inoculum concentration influenced the biodegradability of cyanamide.

 

But, as rapid degradation of cyanamide could clearly be demonstrated under environmentally realistic conditions in two aerobic water/sediment model systems (see IUCLID section 5.2.2) and in soil (see IUCLID section 5.2.3), the substance can thus be considered as rapidly degradable according to the CLP Regulation (EC) No 1272/2008, Annex I sections 4.1.2.9.2 and 4.1.2.9.3. Therefore cyanamide (and calcium cyanamide in a read-across approach) were concluded to be "readily biodegradable".