Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2012 to 29 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 301B, CO2 evolution test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
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: Following pre-study solubility work it was decided that the recommendations of the International Standards Organisation (ISO, 1995) should be followed to prepare the test item solution. Accordingly, an amount of test item (1005 mg) was dissolved in 10 mL of chloroform with the aid of ultrasonication for 5 minutes to give a 1005 mg/10 mL solvent stock solution. An aliquot (400 µL) of the stock solution was dispersed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high sheer mixing (approximately 7500 rpm, 5 minutes) prior to addition to the test vessel containing inoculated mineral medium. An aliquot (51.4 mL) of sodium benzoate stock solution (1000mg/L) was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 13.4 mg test item/L plus 17.1 mg sodium benzoate/L equivalent to a total 20 mg carbon/L. The volumetric flask containing the solvent solution was inverted several times to ensure homogeneity of the solution. Using this method the test item is evenly distributed throughout the test medium and the surface area of the test item exposed to the test organisms is increased thereby increasing the potential for biodegradation..
- Inoculum blank: Inoculated mineral medium containing glass filter paper pre-washed with chloroform, which was allowed to evaporate before use.
- Procedure control: Reference item (sodium benzoate) in inoculate medium plus glass filter paper pre-washed with chloroform, which was allowed to evaporate, to give a final concentration of 10 mg carbon/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Cloudy light brown dispersion containing broken up pieces of filter paper. No undissolved test or reference item visible.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum: Aeration stage of sewage treatment plant which treats predominantly domestic sewage.
- Storage conditions: Continuously aerated at 21°C
- Storage length: Used on day of collection
- Preparation of inoculum for exposure: Washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may be present. Test vessels containing 2400 mL mineral medium and 22.5 mL inoculum were aerated with CO2 free air overnight prior to the addition of the test and reference items. The volume in all vessels was finally adjusted to 3 litres.
- Concentration of sludge: 2.9g suspended solids/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
28 d
Post exposure observation period:
No data reported
Hardness:
No data reported
Test temperature:
22 ± 2°C
pH:
7.4 to 7.8
Dissolved oxygen:
No data reported.
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentration: 13.4 mg test item/L mg equivalent to 10 mg Carbon/L. As the test item contained 50% of the active ingredient then nominal concentration of active test item is 6.7 mg active ingredient/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 5L test culture vessel containing 3L of test solution
- Aeration: Test vessels were sealed and CO2-free air was bubbled through the solutions at rates of 30 to 100 mL/minute/vessel and stirred continuously with a magnetic stirrer. CO2-free air was produced by passing compressed air through a glass column containing self-indicating lime soda.
- No. of vessels per concentration (replicates): One
- No. of vessels per control (replicates): Two for both inoculated control and reference item control.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD guideline mineral medium containing KH2PO4 (0.0850g/L), K2HPO4 (0.2175 g/L), Na2HPO4.2H2O (0.3340 g/L), NH4Cl (0.005 g/L), CaCl2 (0.0255 g/L), MgSO4.7H2O (0.0225 g/L) and FeCl3 (0.00025 g/L).
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH measured at Days 0 and 28

OTHER TEST CONDITIONS
- Adjustment of pH: If necessary, the pH was adjusted to 7.4 ± 0.2 using dilute hydrochloric acid or sodium hydroxide solution prior to aeration of the mineral medium
- Photoperiod: Total darkness

EFFECT PARAMETERS MEASURED: CO2 evolution. CO2 trapped in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH solutions in purified degassed water. 2 mL samples were taken from the first CO2 absorber vessels on days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and analysed for CO2 immediately using IC channel of TOC analyser. Second absorber vessels were sampled on days 0 and 29.


TEST CONCENTRATIONS
Limit test with single test item concentration of 13.4 mg test item/L. As the test item contained 50% of the active test item then nominal concentration of active test item is 6.7 mg active test item/L.
Reference substance (positive control):
yes
Remarks:
Sodium Benzoate
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
6.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Based on the test item containing 50% of active material.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
13.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Carbon content
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Results: The toxicity to micro-organisms result was taken from the toxicity control of a ready biodegradability test. At the only concentration tested (6.7 mg active ingredient/L), the toxicity control (test item plus sodium benzoate) obtained ≥ 25% degradation by Day 14 (actual 44%) and therefore the active ingredient can be considered to be non-inhibitory to the micro-organisms tested.
Results with reference substance (positive control):
The reference substance (sodium benzoate) attained 96% degradation after 14 days and 91% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to sampling /analytical variation. The slight decrease in degradation between days 14 and 28 was also considered to be due to sampling/analytical variation (see table below).
Reported statistics and error estimates:
No data reported

Table 1. Percentage Biodegradation Values

Day

 

Percentage Biodegradation

Sodium Benzoate Procedure Control

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

2

61

17

6

83

27

8

80

30

10

108

43

14

96

44

21

101

55

28

89

59

29*

91

56

*             Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Validity criteria fulfilled:
yes
Remarks:
The total CO2 evolution in the inoculum control was < 40 mg/L (actual 24.28 mg/L). The IC content of the test item in the mineral medium at test start was < 5% (actual 0%). The difference in CO2 production at test end between replicates was < 20%.
Conclusions:
The No Observed Effect Concentration (NOEC) for aquatic micro-organisms is 6.7 mg active ingredient/L.
Executive summary:

The No Observed Effect Concentration (NOEC) for aquatic micro-organisms is 6.7 mg active ingredient/L. The toxicity of the test item to activated sewage sludge was taken from the toxicity control of a GLP-compliant ready biodegradability CO2 evolution test following OECD guideline 301B (Harlan 2012). The study is considered reliable and relevant for use for this endpoint.

Description of key information

The No Observed Effect Concentration (NOEC) for aquatic micro-organisms is 6.7 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
6.7 mg/L

Additional information

The toxicity of the test item to activated sewage sludge was taken from the toxicity control of a GLP-compliant ready biodegradability CO2 evolution test following OECD guideline 301B (Harlan 2013). In the toxicity control, the biodegradation rate of a reference substance known to be readily biodegradable is followed in the presence of the test item. The test item did not exhibit any inhibitory effects on the biodegradation rate of the reference substance, indicating that it was not toxic to the micro-organisms at the concentration tested.