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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-08-18 to 2014-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The inhibition control in standardised biodegradation tests is considered to be an acceptable alternative for the standard OECD TG 209 result
Justification for type of information:
The inhibition control in standardised biodegradation tests is considered to be an acceptable alternative for the standard OECD TG 209 result.
Qualifier:
according to
Guideline:
other: EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Principles of method if other than guideline:
The toxicity to micro-organisms has been evaluated in the ready biodegradability test. This result will be used to evaluate the hazard to micro-organisms in an sewage treatment plant.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
SAMPLING
- Sampling frequency: ten samples were taken within the 28 days
- Sampling method: From each front scrubber flask, ten samples were taken in order to determine the emitted CO2 (on days 0, 2, 4, 7, 9, 11, 14, 18, 23, and 29). The sample volume was 1 mL.
On day 28, 5 mL 2 M HCl were added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.
Details on test solutions:
- Stock solutions:
Solution a
Potassium dihydrogen phosphate (KH2PO4) 8.5 g
Di-potassium hydrogen phosphate (K2HPO4) 21.75 g
Di-sodium hydrogen phosphate dihydrate (Na2HPO4*2H2O) 33.4 g
Ammonium chloride (NH4Cl) 0.5 g
H2O demin. ad 1000 mL
The pH was adjusted to 7.4 +/- 0.1.

Solution b
Calcium chloride dihydrate (CaCl2*2H2O) 36.4 g
H2O demin. ad 1000 mL

Solution c
Magnesium sulfate heptahydrate (MgSO4*7H2O) 22.5 g
H2O demin. ad 1000 mL

Solution d
Iron(III) chloride hexahydrate (FeCl3*6H2O) 0.25 g
Di-sodium ethylenediamine tetraacetate dihydrate (Na2EDTA*2H2O) 0.4 g
H2O demin. ad 1000 mL

- Test medium:
Solution a 10 mL
Solution b 1 mL
Solution c 1 mL
Solution d 1 mL
H2O demin. ad 1000 mL
The medium was freshly prepared.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2, containing mineral medium and inoculum
- Abiotic sterile control: 1, containing test item, mineral medium and HgCl2
- Toxicity control: 1, containing test item, positive control, mineral medium and in-oculum
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant.
- Pretreatment: The sludge was filtered, washed with tap water twice, then washed with and resuspended in the test medium. It was then aerated until use. The dry matter was determined with 4220 mg suspended solids/litre.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Test temperature:
21.6-23.5
pH:
7.3 at the end of the test.
Reference substance (positive control):
yes
Remarks:
Aniline
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
27 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: respiration rate
Details on results:
• The test item nicotine is considered as “readily biodegradable“.
• The degree of biodegradation reached 71% after 28 days.
• The 10-day-window began on day 3, at its end, 72% were reached, surpassing the pass level of 60% given in the OECD guideline.
• Abiotic degradation was not observed.

As degradation in the toxicity flask was 70% after 14 days, the test item can be stated as "not toxic towards the inoculum in a concentration of 27 mg/L.
Results with reference substance (positive control):
See table at "Any other information on results incl. tables". Degradation of positive control was >60% within 7 days.

 Day  Positive control 1  Positive Control 2 Positive Control Mean   Test 1 Test 2 Test Mean  Abiotic control  Toxicity control 
 2  0.1  2.7  1.4  0.2  0.8  0.5  -0.1  0.1
 4  25.6  24.4  25.0  31.0  40.8  35.9  0.1  30.7
 7  69.3  73.5  71.4  64.3  66.4  65.3  -0.5  67.5
 9  75.1  78.6  76.8  74.3  75.4  74.8  -1.1  74.1
 11  73.9  71.5  72.7  70.7  70.2  70.4  -0.9  68.6
 14  72.4  77.8  75.1  73.0  73.6  73.3  -1.3  69.5
 18 76.7   80.1  78.4  74.9  78.6  76.7  -1.8  80.7
 23  82.9  88.2  85.6  78.4  80.3  79.3  -2.3  79.6
 29  72.0  80.9  76.4  70.7  70.8  70.8  -3.9  73.1
Validity criteria fulfilled:
yes
Remarks:
All validity criteria were met. Degradation of the positive control was 71 % after seven days.
Conclusions:
Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation was not observed. Both replicates of the test item showed very good correspondence.
The validity criteria were met and the criterion for ready biodegradability was fulfilled.
If degradation in the toxicity flask is below 25% after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 70% after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 27 mg/L”.

No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.
Executive summary:

The test item nicotine was tested using a concentration of at least 20 mg organic carbon/L (corresponding to 27.0 mg nicotine/L) in test medium following OECD 301B and EU-Method C.4-C.

 

Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L). The test was left running for 28 days.

All validity criteria were met. Degradation of the positive control was 71% after seven days.

 

The following data were determined for the test item nicotine:

10-day-window: day 3 – 13
degradation at the end of 10-day-window: 72%

degradation at the end of the test: 71%

pass level following guideline: 60% at the end of 10-day-window

No inhibition of the degradation of the reference substance was observed in the toxicity control of this test in the presence of 27 mg/L.

Description of key information

One key study of high quality is available (Klimisch 1). The study is GLP compliant and appropriate for risk assessment, therefore no further testing is needed.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
27 mg/L

Additional information