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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
The concentration and stability of the test substance in the sludge-samples is not determined analytically. The data about the identity and stability of the test substance are at the responsibility of the sponsor (see Annex I of study report).
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
The concentration and stability of the test substance in the sludge-samples is not determined analytically. The data about the identity and stability of the test substance are at the responsibility of the sponsor (see Annex I of study report).
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A solution was prepared from "1-NITROGUANIDINE" by diluting 687.4 mg test substance in 1000 mL tap water, corresponding to 529.3 mg dry weight/L. This stock solution was used for the preparation of the different incubation mixtures.
- For the actual amounts of each sample see Table 1.

Preparation of the positive control substance solution:
- A stock solution of 3,5-dichlorophenol (nominally 0.5 g/L) was prepared by dissolving 50.3 mg of the substance in 100 mL of deionised water.
- Three concentrations, nominally from 5 to 30 mg/L, were prepared as described in Table 1.
- No NaOH was used to increase the solubility of dichlorophenol, therefore no adjustment of the pH was necessary.
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.
- A sample of activated sludge was collected on the day of 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.
- 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 re-weighing 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 (final concentration of 1.6 g dry weight per litre in the samples). This suspension was used as inoculum for the samples. The inoculum was aerated until use.
- After mixing the inoculum with synthetic sewage solution and appropriate dilutions of control or test substance solutions the samples were aerated for a contact time of 3 hours at 20 °C.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
10.5 min
Remarks on exposure duration:
at 20 °C (incubation time)
Hardness:
Not reportes
Test temperature:
20 °C
pH:
The pH of the incubation mixtures with the test substance after the 3 hour incubation was between 8.3 and 8.4, the pH of the negative control samples was 8.4 and 8.5. The pH of the standardised activated sludge was 7.8.
The test substance did not influence the pH.
Dissolved oxygen:
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.
Salinity:
not applicable
Nominal and measured concentrations:
Nominal: 7.7, 19.1, 47.6, 120.7 and 300.6 mg/L
Details on test conditions:
TEST SYSTEM
- All incubation mixtures were prepared by adding 200 mL of microbial inoculum and 16 mL of concentrated synthetic sewage solution (see 4.7) to 284 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
- Aeration: 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
- No. of vessels per concentration (replicates): 5
- No. of vessels per positive control (replicates): 3
- No. of vessels per negative control (replicates): 2
- Biomass loading rate: 1.6 g dry weight per litre
- Negative controls were run before the first and after the last test substance sample.
- 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 %.




EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.

Measurement of pH:
- pH was determined with a pH-meter in the standardised microbial inoculum before the test and in each sample at the end of the incubation and aeration.

Temperature registration:
- The test was performed in a temperature controlled room with automatic temperature registration.
- The temperature inside the samples was measured with a mercury thermometer.



TEST CONCENTRATIONS
- The highest concentration of the test substance corresponds to the scheme given in the guidelines.
- The spacing between the concentrations of 2.5 is within the permitted range of 'not more than 3.2'.
- The amount of concentrations tested is in agreement with the guidelines which require 'at least 5'.
- The concentrations of 3,5-dichlorophenol were set around the expected EC50 of 5 to 30 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 300 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: No confidence limits can be calculated.
Key result
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
Remarks on result:
other: No confidence limits can be calculated.
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
Remarks on result:
other: No confidence limits can be calculated.
Details on results:
- The test substance did not inhibit the respiration of the activated sludge. Even a small enhancement of the respiration rate could be observed which may be due to fermentation of the test substance by the bacteria.
- The respiration rates of all samples with the test substance were in the range of 94.1 to 105.3 % of the control value.

The dose - response diagram of the test substance is presented in Figure 1.
Results with reference substance (positive control):
Test conditions:
- The amount of dry substance in the standardised activated sludge was 4 g/L
- The pH of the standardised activated sludge was 7.8.
- The temperature in the samples was 20 °C.
- 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 = 8.4 mg/L EC50 = 18.3 mg/L EC80 = 39.7 mg/L

Figure 2 shows the graph of the inhibition data of 3,5-dichlorophenol.
Validity criteria fulfilled:
yes
Remarks:
1) The respiration rates of the two negative control samples were within 15 % of each other. Actual values: ± 4.7 %. 2) The EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L. Actual EC50: 18.3 mg/L.
Conclusions:
The test substance did not inhibit the respiration of the activated sludge. Nitroguanidine is not toxic to aerobic microorganisms in sewage sludge.
Executive summary:

The study was performed to estimate possible effects of nitroguanidine 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" and the EU- COUNCIL REGULATION (EC) No 440/2008, using activated sludge from a sewage treatment plant treating predominantly domestic sewage.

Five concentrations of the test substance (7.7, 19.1, 47.6, 120.7 and 300.6 mg dry weight/L) were tested versus two negative controls (tap water). As positive control substance 3,5-dichlorophenol was used and tested in three concentrations (5.0, 12.3 and 30.2 mg/L).

The test substance did not inhibit the respiration of the activated sludge.

The respiration rates of all samples with the test substance were in the range of 94.1 to 112.6 % of the control value.

The following EC-values for nitroguanidine were obtained (values are rounded off to two significant figures):

EC20: greater than 300 mg/L

EC50: greater than 300 mg/L

EC80: greater than 300 mg/L

Description of key information

To test the toxicity of nitroguanidine towards microorganisms, a test according to OECD 209 (activated sludge respiration inhibition test) was conducted. At five concentrations up to 300.6 mg dry weight/L. The test substance did not inhibit the respiration of the activated sludge at any concentration tested (NOEC ≥ 300 mg/L). Therefore, it can be concluded that nitroguanidine is not toxic to aerobic microorganisms in sewage sludge.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
300 mg/L

Additional information

To test the toxicity of nitroguanidine towards microorganisms, a test was performed according to the OECD Guideline for Testing of Chemicals 209 "Activated Sludge, Respiration Inhibition Test" and the EU- COUNCIL REGULATION (EC) No 440/2008, using activated sludge from a sewage treatment plant treating predominantly domestic sewage.

Five concentrations of the test substance (7.7, 19.1, 47.6, 120.7 and 300.6 mg dry weight/L) were tested versus two negative controls (tap water). As positive control substance 3,5-dichlorophenol was used and tested in three concentrations (5.0, 12.3 and 30.2 mg/L).

The test substance did not inhibit the respiration of the activated sludge.

The respiration rates of all samples with the test substance were in the range of 94.1 to 112.6 % of the control value.

The following EC-values for nitroguanidine were obtained (values are rounded off to two significant figures):

EC20: greater than 300 mg/L

EC50: greater than 300 mg/L

EC80: greater than 300 mg/L

It can be concluded that nitroguanidine is not toxic to aerobic microorganisms in sewage sludge. Accordingly, the highest concentration of 300 mg/L is adopted in lieu of a NOEC.