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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:
Study was carried out between 22 and 23 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
14 June 2010
Analytical monitoring:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low aqueous solubility of the mixed ash, a stock solution was not prepared. The appropriate mass of mixed ash was added directly to the test flasks.
- Controls: culture control
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sludge was obtained from Totnes Sewage Treatment Works, Totnes, Devon, UK, three days prior to the exposure of test and reference substances. The activated sludge was concentrated by settling, after which the supernatant was decanted and the sludge was washed with tap water. The washed, settled sludge was fed (50 mL of OECD synthetic sewage feed per litre of sludge per day) and aerated at room temperature until used in the test. The total filterable solids concentration, determined on the day of the test, was 6289 mg/L.

The synthetic sewage feed (according to the OECD guideline):
- 16 g of peptone
- 11 g of meat extract
- 3.0 g of urea
- 0.7 g of NaCl
- 0.4 g of CaCl2.2H2O
- 0.2 g of MgSO4.7H2O
- 2.8 g of K2HPO4
per litre of hot tap water
Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
Not available.
Test temperature:
20 ± 2˚C (nominal)
pH:
7.7
Dissolved oxygen:
Not available.
Salinity:
Not available.
Nominal and measured concentrations:
Nominal concentrations 1.0, 3.2, 10, 32 and 100 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: flask
- Type: open
- Material, size, headspace, fill volume: glass, 500 mL
- Aeration: Yes
- No. of vessels per concentration (replicates): one for the Mixed ash and one for the reference substance (no replicates). An additional abiotic reference substance flask at concentration 100 mg/L without replicates (table 1).
- No. of vessels per control (replicates): two

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis (RO) water.
- Particulate matter: 1600 mg/L in the test flasks (activated sludge).
- Intervals of water quality measurement: pH measurement at the start and end of the test. The temperatures of the flask contents were measured at the end.

OTHER TEST CONDITIONS
- Adjustment of pH: only for the activated sludge prior test from 5.9 to 7.7 with 2 M NaOH.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mg O2/Lh at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: none
- Test concentrations: 1.0, 3.2, 10, 32 and 100 mg/L

The exact quantities of constituents are given in Table 1.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 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:
NOEC
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The 20% and 80% effect concentrations (EC20 and EC80) are defined as the concentrations, estimated from the data obtained, resulting in 20 and 80% reduction respectively in the respiration rate of activated sludge within the period of the test, therefore, for mixed ash:

EC20 >100 mg L-1
EC80 >100 mg L-1
Results with reference substance (positive control):
- Results with reference substance valid? Yes; the 3 hour EC50 value for the reference substance was estimated to be 12 mg/L (Fig 1). According to the OECD Guideline, this is within the expected normal range of 5 to 30 mg/L indicating that the sludge was responding normally and confirming the viability of the sludge organisms.
Reported statistics and error estimates:
The respiration rates in the two control flasks were within 15% of each other.

The 10% effect concentration (EC10) is defined as the concentration, estimated from the data obtained, resulting in a 10% reduction in the respiration rate of activated sludge within the period of the test. Percentage inhibition less than or equal to 10% was within the expected experimental variability of the test and was considered not to be an effect of mixed ash. The no observed effect concentration (NOEC) is therefore equivalent to the EC10 in this test.

% Inhibition = [1 -[Respiration rate of test flask/Mean resp. rate of control flask]]x100

TABLE 2     Inhibition Results

Test substance

Nominal test concentration

pH of test flasks

 Respiration rate

 

Percentage inhibitiona

 

Start

 

 End

 

(mg L-1)

(mg O2L‑1h-1)

 

Control

Control

7.6

6.6

61.2

-

3,5-DCPc

100 (abiotic)

7.7

7.1

-

 -

3,5-DCP

100

7.6

7.5

5.2

92

3,5-DCP

32

7.5

7.8

8.1

87

3,5-DCP

10

7.5

7.7

35.8

42

3,5-DCP

3.2

7.5

7.7

44.0

29

Control

Control

7.3

6.5

62.2

 -

Mixed ash

100

7.5

6.4

74.1

<10

Mixed ash

32

7.4

6.5

74.1

<10

Mixed ash

10

7.3

6.4

63.5

<10

Mixed ash

3.2

7.3

6.4

60.7

<10

Mixed ash

1.0

7.3

6.4

63.7

<10

a Calculated using mean control respiration rate = 61.7 mg O2/Lh

b 3,5-dichlorophenol

Validity criteria fulfilled:
yes
Conclusions:
The effect concentration (EC50) for respiration of activated sludge was > 100 mg/L
Executive summary:

Toxicity of ash to micro-organisms was determined in a GLP compliant laboratory study according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test). Because of low water solubility, ash was added directly to the test flasks without preparing test solutions. Additionally, reverse osmosis water, activated sludge and synthetic sludge were included in the test flasks. A reference substance was 3,5-dichlorophenol. The respiration rate of each culture was measured with microcathode oxygen electrodes after three hours exposure at the end of the test and compared with the mean respiration rate of the two control cultures. Finally, the EC50value was estimated by Strathkelvin ASR System Data Analysis software, Version 3.0.5.0. The EC50value was found to be >100 mg/L.

Description of key information

Toxicity of Ash to micro-organisms was determined in a GLP compliant laboratory study according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test). The EC50 (3h) was >100.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

Due to low water solubility, Ash was used in the test as solid without preparing a stock solution.