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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2010-03-17 to 2010-08-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
Regulation No 440/2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted 1984-04-04
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
no analytical monitoring
Vehicle:
no
Details on test solutions:
The test substance was not enough soluble in water to prepare a stock solution. Therefore suspensions of the test substance were prepared by stirring the appropriate amounts of test substance in the test vessels with 100 mL tap water for 2 days prior to the start of the test. The stock suspensions were used without filtration.
All incubation mixtures were prepared by adding 200 mL of microbial inoculum and 200 mL of synthetic sewage solution to 100 mL of tap water and/or the appropriate amount of the test or control substance in glass beakers with a nominal volume of 1000 mL. The samples consisted of two negative controls (one before and one after all the test substance samples), five concentrations of calcium cyanamide, and three concentrations of the positive control (3,5-dichlorophenol).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
For the preparation of the microbial inoculum activated sludge was used. The sludge was collected from one of the return sludge channels of the sewage treatment plant in Baden near Vienna (Zubringerstraße, A-2500 Baden). Baden is a spa with only a small amount of industry and the waste water of the sewage treatment plant is predominantly domestic.

Preparation of the microbial inoculum
A sample of activated sludge was collected two days before the test. The container for transportation was only maximally 2/3 filled in order to maintain the contact with air. At arrival in the laboratory (less than 1 hour after sample collection), the supernatant over the sludge was decanted, the sludge was diluted with tap water and aerated by means of compressed air. 50 mL of synthetic sewage concentrate were added per litre each day. On the day of the test the sludge was allowed to sediment and the supernatant was decanted. The concentration of suspended solids was determined by filtering 3 mL of the sample through a pre-dried and pre-weighed glass microfibre filter. After drying at 110 °C and reweighing the amount of solids was calculated. On the basis of this calculation the concentration of suspended solids was adjusted to 4 g/L with tap water. This suspension was used as inoculum for the samples. The inoculum was aerated until use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
none
Hardness:
no data
Test temperature:
The temperature in the samples was 20 °C.
pH:
The pH of the standardised activated sludge was 7.91.
Dissolved oxygen:
Negative control: O2 at begin of study: 5.54 mg/L
Salinity:
not applicable
Nominal and measured concentrations:
Nominal: 4.7, 9.4, 18.8, 37.5, 75.0, 150.0 and 300 mg/L
Details on test conditions:
After mixing, the samples were aerated at a flow rate of 0.7 L/min using oil free compressed air and a Pasteur-pipette as aeration device. The incubation of the individual samples was started at intervals of 12-13 min and each sample was aerated for 3 hours at 20 °C. At the end of the incubation time the pH was determined and aliquots of the samples were transferred to measuring bottles and oxygen consumption was recorded.

Measurement of dissolved oxygen concentration
The oxygen concentration was determined with a DO-meter "OXI Level 2" and an oxygen electrode (Wissenschaftlich-technische Werkstätten G.m.b.H., Dr. rer.nat. K. Slevogt, D-82362 Weilheim i. OB, Trifthofstr. 57a). The readout was set to 100 % in water-saturated air.

Measurement of respiration rates
Respiration rates (in mg O2.L-1.h-1 ) were determined in aliquots of the incubation mixtures. Bottles with narrow neck and a volume of about 100 mL were filled with the sample and positioned on a magnetic stirrer. Then the oxygen electrode (equipped with a magnetic stirring wheel) was inserted in a way that no air remained in the bottle and that the electrode sealed the bottle neck to avoid contact of the sample with the atmosphere. The decline of oxygen concentration was measured by recording the concentrations of oxygen at various times.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
ca. 30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
ca. 89 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 46 to 130 m/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The respiration rates of all samples with the test substance were in the range of 69.7 to 102.9 % of the control value. The pH of the incubation mixtures after the 3 hour incubation was between 8.25 and 8.55, the pH of the negative control samples was 8.26 and 8.34. The test substance did not influence the pH.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L. The actual values of the EC20, EC50 and EC80 for 3,5-dichlorophenol were:
EC20 = 5.6 mg/L
EC50 = 12.1 mg/L
EC80 = 26.2 mg/L
Reported statistics and error estimates:
The concentrations of the test substance around 50 % inhibition (if such data exist) and the concentrations of the positive control are converted to log10. These values and the corresponding percentage of inhibition are subjected to linear regression analysis. These analyses are used to obtain the regression coefficients and the respective EC10, EC20, EC50 and EC80 values.
Validity criteria fulfilled:
yes
Conclusions:
In a activated sludge respiratory inhibition test a EC50 value of >300 mg/L was determined after an incubation time of 3 h. The EC10 was calculated to be 30 mg/L.
Executive summary:

Objective of the study

The study was performed to estimate possible effects of calcium cyanamide on aerobic microbial sewage treatment plants. The test was performed according to the OECD Guideline for Testing of Chemicals 209 "Activated Sludge, Respiration Inhibition Test" (April 1984) and the EU-COUNCIL REGULATION (EC) No 440/2008, using activated sludge from a sewage treatment plant treating predominantly domestic sewage.

Test design

Seven concentrations of the test substance (4.8, 9.0, 18.2, 37.6, 75.2, 151.2 and 300.2 mg/L) were tested versus two negative controls (tap water). As the test substance was not enough soluble in water to prepare a stock solution, suspensions of the test substance in water were prepared and stirred for 46 h. As positive control substance 3,5-dichlorophenol was used and tested in three concentrations (5.1, 12.3 and 30.4 mg/L). After incubation the respiration rates were determined in closed bottles using an oxygen sensitive electrode. The inhibition of respiration was calculated from the respiration rates using the mean value of the negative controls as 100 %.

Results

The test results fulfilled the criteria for validity:

- the respiration rates of the two negative control samples were within 15 % of each other. Actual values: ± 4.7 %.

- the EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L. Actual EC50: 12.1 mg/L.

The respiration rates of all samples with the test substance were in the range of 69.7 to 102.9 % of the control value. The following EC-values for calcium cyanamide were obtained (values rounded off to two significant figures):

EC10: 30 mg/L, no confidence limits can be calculated

EC20: 89 mg/L, confidence limits: 46 to 130 mg/L

EC50: > 300 mg/L, no confidence limits can be calculated

EC80: > 300 mg/L, no confidence limits can be calculated

Endpoint:
toxicity to microorganisms, other
Remarks:
growth inhibition
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Guideline of the Umweltbundesamt ”Bewertung wassergefährdender Stoffe
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
No sampling
Vehicle:
no
Details on test solutions:
Not indicated
Test organisms (species):
Pseudomonas putida
Details on inoculum:
Not indicated
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
19 h
Post exposure observation period:
No post exposure observation period
Hardness:
Not
Test temperature:
25°C
pH:
Not indicated
Dissolved oxygen:
Not indicated
Salinity:
freshwater
Nominal and measured concentrations:
100, 180, 320, 561 and 1002 mg/L of 49 % (w/w) aqueous solution of cyanamide.
Details on test conditions:
In order to determine the percent growth inhibition, at the end of the study the optical density was measured in the nutrient solutions with the 49 % (w/w) aqueous solution of cyanamide and in the control solution without test substance.
Reference substance (positive control):
no
Duration:
19 h
Dose descriptor:
other: TT: toxicity threshold (the concentration of the test substance at which its inhibitory action starts)
Effect conc.:
157 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Duration:
19 h
Dose descriptor:
EC50
Effect conc.:
283 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Duration:
19 h
Dose descriptor:
NOEC
Effect conc.:
88 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Details on results:
An inhibition of the growth of Pseudomonas putida was first observed at a concentration of 320 mg/L, where the growth was reduced for 10.3 % in comparison to the control. The highest dose (1002 mg/L) resulted in an inhibition of 90 % .
Results with reference substance (positive control):
No positive control
Reported statistics and error estimates:
Not indicated

Effects of 49% (w/w) aqueous solution of cyanamide on biological methods for sewage treatment:

49% (w/w) aqueous solution

of cyanamide (mg/L)

Pure active ingredient cyanamide (mg/L)

Growth inhibition (%)

0

0

-

100

49

0

180

88

0.6

320

157

10.3

561

275

69.1

1002

491

90.0

Validity criteria fulfilled:
not specified
Conclusions:
The toxicity threshold (19 hours) was found to be 320 mg product (49 % (w/w) aqueous solution of cyanamide/L), corresponding to 157 mg as/L. The EC50 (19 hours) was calculated to be 577 mg product/L, corresponding to 283 mg as/L.The NOEC was determined to be 180 mg product/L, corresponding to 88 mg as/L. Therefore, it can be concluded that cyanamide poses a very low risk to biological methods for sewage treatment.
Due to its fast dissipation in soil and water, cyanamide is not expected to reach water processing facilities at high concentrations.
Executive summary:

The influence of aqueous hydrogen cyanamide solution on the growth and degradation performance was conducted, using Pseudomonas putida as model organisms in fresh water. A 49 % (w/w) aqueous solution of cyanamide at concentrations of 100, 180, 320, 561 and 1002 mg/L and cultures of Pseudomonas putida were incubated for 19 hours at 25 °C. In order to determine the percent growth inhibition, at the end of the study the optical density was measured in the nutrient solutions with the 49 % (w/w) aqueous solution of cyanamide and in the control solution without test substance.

An inhibition of the growth of Pseudomonas putida was first observed at a concentration of 320 mg/L, where the growth was reduced for 10.3 % in comparison to the control. The highest dose (1002 mg/L) resulted in an inhibition of 90 %.

The toxicity threshold (19 hours) was found to be 320 mg product (49 % (w/w) aqueous solution of cyanamide/L), corresponding to 157 mg as/L. The EC50 (19 hours) was calculated to be 577 mg product/L, corresponding to 283 mg as/L. The NOEC was determined to be 180 mg product/L, corresponding to 88 mg as/L.

Therefore, it can be concluded that cyanamide poses a very low risk to biological methods for sewage treatment.

Due to its fast dissipation in soil and water, cyanamide is not expected to reach water processing facilities at high concentrations.

Endpoint:
toxicity to microorganisms, other
Remarks:
growth inhibition
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
Duration:
19 h
Dose descriptor:
other: TT: toxicity threshold (the concentration of the test substance at which its inhibitory action starts)
Effect conc.:
157 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Duration:
19 h
Dose descriptor:
EC50
Effect conc.:
283 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Duration:
19 h
Dose descriptor:
NOEC
Effect conc.:
88 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Details on results:
An inhibition of the growth of Pseudomonas putida was first observed at a concentration of 320 mg/L, where the growth was reduced for 10.3 % in comparison to the control. The highest dose (1002 mg/L) resulted in an inhibition of 90 % .
Results with reference substance (positive control):
No positive control
Reported statistics and error estimates:
Not indicated

Effects of 49% (w/w) aqueous solution of cyanamide on biological methods for sewage treatment:

49% (w/w) aqueous solution

of cyanamide (mg/L)

Pure active ingredient cyanamide (mg/L)

Growth inhibition (%)

0

0

-

100

49

0

180

88

0.6

320

157

10.3

561

275

69.1

1002

491

90.0

Validity criteria fulfilled:
not specified
Conclusions:
The toxicity threshold (19 hours) was found to be 320 mg product (49 % (w/w) aqueous solution of cyanamide/L), corresponding to 157 mg as/L. The EC50 (19 hours) was calculated to be 577 mg product/L, corresponding to 283 mg as/L.The NOEC was determined to be 180 mg product/L, corresponding to 88 mg as/L. Therefore, it can be concluded that cyanamide poses a very low risk to biological methods for sewage treatment.
Due to its fast dissipation in soil and water, cyanamide is not expected to reach water processing facilities at high concentrations.

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 influence of aqueous hydrogen cyanamide solution on the growth and degradation performance was conducted, using Pseudomonas putida as model organisms in fresh water. A 49 % (w/w) aqueous solution of cyanamide at concentrations of 100, 180, 320, 561 and 1002 mg/L and cultures of Pseudomonas putida were incubated for 19 hours at 25 °C. In order to determine the percent growth inhibition, at the end of the study the optical density was measured in the nutrient solutions with the 49 % (w/w) aqueous solution of cyanamide and in the control solution without test substance.

An inhibition of the growth of Pseudomonas putida was first observed at a concentration of 320 mg/L, where the growth was reduced for 10.3 % in comparison to the control. The highest dose (1002 mg/L) resulted in an inhibition of 90 %.

The toxicity threshold (19 hours) was found to be 320 mg product (49 % (w/w) aqueous solution of cyanamide/L), corresponding to 157 mg as/L. The EC50 (19 hours) was calculated to be 577 mg product/L, corresponding to 283 mg as/L. The NOEC was determined to be 180 mg product/L, corresponding to 88 mg as/L.

Therefore, it can be concluded that cyanamide poses a very low risk to biological methods for sewage treatment.

Due to its fast dissipation in soil and water, cyanamide is not expected to reach water processing facilities at high concentrations.

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

For detailled description where read across is used/recommended and where it is preferrable to refain from read across, please see section 13.2 "read across justification for environmental endpoints" and "Scientific rationale for not using cyanamide as read-across substance for calcium cyanamide on toxicological endpoints"

Description of key information

In an activated sludge respiratory inhibition test with calcium cyanamide, technical grade (Kalkstickstoff) an EC50 value of >300 mg/L was determined after an incubation time of 3 h. The EC10 was calculated to be 30 mg/L. The sample tested contains 69.3 % calcium cyanamide. Re-calculation to 100 % calcium cyanamide results in an EC50 of > 109.3 mg/L and an EC10 of 10.9 mg/L.

In addition, information for the read-across substance cyanamide is available:
The toxicity of cyanamide to microorganisms was examined in one available study using Pseudomonas putida as model organisms in fresh water. The toxicity threshold (19 hours) was found to be 157 mg ai/L. The EC50 (19 hours) 283 mg as/L. The NOEC was determined to be 180 mg product/L, corresponding to 88 mg as/L.

The more sensitive effect value obtained for calcium cyanamide in a standard study are used for risk assessment and determination of the PNEC(STP) (effect values expressed as mg cyanamide/L).

Key value for chemical safety assessment

EC50 for microorganisms:
109.3 mg/L
EC10 or NOEC for microorganisms:
10.9 mg/L

Additional information

Objective of the study with calcium cyanamide:

The study was performed to estimate possible effects of calcium cyanamide on aerobic microbial sewage treatment plants. The test was performed according to the OECD Guideline for Testing of Chemicals 209 "Activated Sludge, Respiration Inhibition Test" (April 1984) and the EU-COUNCIL REGULATION (EC) No 440/2008, using activated sludge from a sewage treatment plant treating predominantly domestic sewage.

Test design

Seven concentrations of the test substance (4.8, 9.0, 18.2, 37.6, 75.2, 151.2 and 300.2 mg/L) were tested versus two negative controls (tap water). As the test substance was not enough soluble in water to prepare a stock solution, suspensions of the test substance in water were prepared and stirred for 46 h. As positive control substance 3,5-dichlorophenol was used and tested in three concentrations (5.1, 12.3 and 30.4 mg/L). After incubation the respiration rates were determined in closed bottles using an oxygen sensitive electrode. The inhibition of respiration was calculated from the respiration rates using the mean value of the negative controls as 100 %.

Results

The test results fulfilled the criteria for validity:

- The respiration rates of the two negative control samples were within 15 % of each other. Actual values: ± 4.7 %.

- The EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L. Actual EC50: 12.1 mg/L.

The respiration rates of all samples with the test substance were in the range of 69.7 to 102.9 % of the control value. The following EC-values for calcium cyanamide were obtained (values rounded off to two significant figures):

EC10: 30 mg/L, no confidence limits can be calculated

EC20: 89 mg/L, confidence limits: 46 to 130 mg/L

EC50: > 300 mg/L, no confidence limits can be calculated

EC80: > 300 mg/L, no confidence limits can be calculated

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 degrade in a soil environment. Exposure of the aquatic environment will be exclusively due to runoff from agricultural fields. As above, cyanamide is the chemical moiety relevant for exposure due to rapid transformation from calcium cyanamide to cyanamide.

(Please note: direct release of the product to surface waters is strictly advised against, thus not a relevant exposure pathway.

For detailled description where read across is used/recommended and where it is preferrable to refain from read across, please see section 13.2 "read across justification for environmental endpoints" and "Scientific rationale for not using cyanamide as read-across substance for calcium cyanamide on toxicological endpoints")

The following information on the read-across substance cyanamide is available:

The toxicity of cyanamide (aqueous 49 % (w/w) hydrogen cyanamide solution) to microorganisms was examined in one available study using Pseudomonas putida as model organisms in fresh water. A 49 % (w/w) aqueous solution of cyanamide at concentrations of 100, 180, 320, 561 and 1002 mg/L were applied to cultures of Pseudomonas putida and incubated for 19 hours at 25 °C. In order to determine the percent growth inhibition, at the end of the study the optical density was measured in the nutrient solutions with the 49 % (w/w) aqueous solution of cyanamide and in the control solution without test substance.

An inhibition of the growth of Pseudomonas putida was first observed at a concentration of 320 mg/L, where the growth was reduced for 10.3 % in comparison to the control. The highest dose (1002 mg/L) resulted in an inhibition of 90 %.

The toxicity threshold (19 hours) was found to be 320 mg product (49 % (w/w) aqueous solution of cyanamide/L), corresponding to 157 mg as/L. The EC50 (19 hours) was calculated to be 577 mg product/L, corresponding to 283 mg as/L. The NOEC was determined to be 180 mg product/L, corresponding to 88 mg as/L.