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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:
7 December 2012 - 23 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
(2010)
Deviations:
yes
Remarks:
The mean blank- control O2 uptake rate was 19 mg O2/g active sludge per h and the guideline value is 20 mg O2/g. Since the deviation is only 1 mg O2/g of AS and the EC50 of the ref. subst. was normal, the test is valid.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(2008)
Deviations:
yes
Remarks:
The mean blank- control O2 uptake rate was 19 mg O2/g active sludge per h and the guideline value is 20 mg O2/g. Since the deviation is only 1 mg O2/g of AS and the EC50 of the ref. subst. was normal, the test is valid.
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
(2007)
Deviations:
yes
Remarks:
The mean blank- control O2 uptake rate was 19 mg O2/g active sludge per h and the guideline value is 20 mg O2/g. Since the deviation is only 1 mg O2/g of AS and the EC50 of the ref. subst. was normal, the test is valid.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
A stock solution of 10 g/L was prepared by adding 5.0 g test substance to 500 ml of Milli-RO water (tap water purified by reverse osmosis. Magnetic stirring was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear slightly yellow stock solution corresponding to the test concentration were then added to the test media.

- Controls:
Blank-control, nitrification control, abiotic control and the highest concentration with a nitrification inhibitor
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure:
The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.4 g/L of sludge, as used for the test). The pH was 7.3 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h
Test temperature:
18 - 20°C
pH:
At the start of the test (before addition of sludge): 7.5 - 7.6.
After the 3 hour exposure period: 7.4 - 8.1
Dissolved oxygen:
At the start of the test the dissolved oxygen concentration was above 60-70% saturation
Nominal and measured concentrations:
Nominal: 0 (control), 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass, open bottles/vessels
- Material, size, headspace, fill volume: Glass, with a capacity of 1000 mL and filled with 500 mL.
- Aeration: yes
- No. of vessels per concentration (replicates): highest concentration was tested in triplicate; lower concentrations only one replicate
- No. of vessels blank-control (replicates): 2
- No. of vessels abiotic-control (replicates): 1
- No. of vessels nitrification-control (replicates): 1
- No. of vessels toxicity-control (replicates): 2
- Loading rate: 1.7 g/L of sludge (suspended solids)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q)

Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Synthetic medium (=sewage feed)
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-Q water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10x
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 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: EC10/NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- The combined limit/range-finding test showed no inhibition, 11 and an average of 6% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. No statistically significant inhibition of the respiration rate of the sludge was recorded at 1000 mg Sodium ferrocyanide per litre.

- In the abiotic control no significant uptake of oxygen was observed

- In the toxicity control a slight heterotrophic inhibition of the respiration rate was observed.

The mean (blank-) control oxygen uptake rate was less than 20 mg oxygen (O2) per gram of activated sludge (dry weight of suspended solids) in one hour (actual value is 19 mg O2 per gram of activated sludge per hour). Since this value represents a deviation of only 1 mg O2 per one gram of activated sludge in relation to the value set on the guideline and the sludge showed normal inhibition to the reference substance, the test was considered to be valid.
Results with reference substance (positive control):
EC50 of 3,5-dichlorophenol was 12 mg/L
Validity criteria fulfilled:
no
Remarks:
The mean (blank-) control O2 uptake rate was less than 20 mg O2/g of activated sludge per hour. This deviation is of only 1 mg O2/g of activated sludge in relation to the required guideline value, and the reference substance effect levels were normal.
Conclusions:
Under the conditions of this present test, Sodium ferrocyanide did not significantly inhibited the respiration rate of waste water (activated sludge bacteria) at concentrations as high as 1000 mg/L. The EC50 exceeded the highest test concentration of 1000 mg/L and and the NOEC was 1000 mg/L.
Executive summary:

The toxicity of Sodium ferrocyanide to microorganisms was determined in a 3 h activated sludge respiration inhibition study carried out according to OECD guideline No. 209 and GLP principles.

In a combined limit/range-finding test, microorganisms in activated sludge were exposed to the test substance at a concentration of 10, 100 and 1000 mg/L under static conditions. Under the chosen test conditions Sodium ferrocyanide did not significantly inhibited the respiration rate of waste water bacteria (activated sludge) at concentrations as high as 1000 mg/L. The EC50 exceeded the highest test concentration of 1000 mg/L and the NOEC was 1000 mg/L.

Although, not all criteria for acceptability of the test were fully met the test was still considered to be valid. The present toxicity study is classified as reliable without restrictions according to the OECD guideline No. 209.

Description of key information

The EC50 for toxicity to microorganisms was determined to be >1000 mg/L, based on read across using sodium ferrocyanide as the source substance.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

Key study (read across):

The toxicity of Sodium ferrocyanide to microorganisms was determined in a 3 h activated sludge respiration inhibition study carried out according to OECD guideline No. 209 and GLP principles.

In a combined limit/range-finding test, microorganisms in activated sludge were exposed to the test substance at a concentration of 10, 100 and 1000 mg/L under static conditions.Under the chosen test conditions Sodium ferrocyanide did not significantly inhibited the respiration rate of waste water bacteria (activated sludge) at concentrations as high as 1000 mg/L. The EC50 exceeded the highest test concentration of 1000 mg/L and the NOEC was 1000 mg/L.

Although, not all criteria for acceptability of the test were fully met the test was still considered to be valid. The present toxicity study is classified as reliable without restrictions according to the OECD guideline No. 209.