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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test conducted according to standard guidelines and GLP-compliant. The validity criteria were not completely met, however this is not believed to have affected the study results. This study is therefore considered to be suitable for use as a key for this endpoint.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
not required
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge was obtained from Totnes Sewage Works, Totnes, Devon UK, one day prior to exposure. Activated sludge was settled and supernatant decanted. Settled sludge was fed with 50 ml OECD synthetic sewage feed per litre of sludge per day and aerated at room temperature. Total filterable solids concentration on day of test was 8574 mg/L, pH was 6.4 and adjusted to 7.1 with 2M sodium hydroxide prior to use
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 +- 2°C after 3 hours aeration
pH:
6.7 - 8.2
Nominal and measured concentrations:
Range finder: 10, 100 and 1000 mg/L (nominal)
Definitive study: 0.2, 0.64, 2.0, 6.4, 20 and 50 mg/L (nominal)
Details on test conditions:
A range finding study was run at 10, 100 and triplicate 1000 mg/L. The estimated EC50 from the range finder was 2.6 mg/L. Ths informed the choice of test concentrations used in the definitive study.

In the definitive study, two sets of exposure periods were run in one day, each set consisted of three test substance concentrations, five reference flasks and four control culture flasks. Five replicates and a control flask were used per test concentration.

Each flask contained an excess of synthetic sewage, sufficient activated sludge to give final solids concentration of 1500 mg/L (with the exception of the abiotic flask), TCA or 3,5-DCP and RO water to give a final volume of 100 mL.

pH was measured at the start and end of the test. Temperature was measured at the end of 3 hours aeration. Respiration rate was measured was measured after 3 hours and compared to the control flask. Rate of oxygen uptake was measured.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
27.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 15.0 - 33.3 mg/L, 95% confidence levels
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
2 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:
LOEC
Effect conc.:
6.4 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: EC20
Effect conc.:
8.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 2.5 - 14.7 mg/L, 95% confidence intervals
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
Average 3 hour EC50: 9.7 mg/L
Validity criteria fulfilled:
no
Remarks:
Criterion of no less than 20 mg oxygen per one gram of activated sludge in an hour was not met by all control flasks. However the consistency of the data shows that slight deviation to the validity criteria has not affected the study results
Conclusions:
The 3-hour EC50 based on respiration rate was 27.9 mg/L. The NOEC was 2 mg/L.
Executive summary:

In a GLP-compliant study following OECD guideline 209, a range-finding test was carried out using 3 concentrations of test item, the highest concentration was tested in triplicate. A definitive test was performed using 6 concentrations (5 replicates per test item) and a control. Results are reported as nominal concentrations. Although the validity criteria were not completely met this is not believed to have affected the study results. This study is therefore considered to be suitable for use for this endpoint. The 3-hour EC50 based on respiration rate was 27.9 mg/L. The NOEC was 2 mg/L.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Tests conducted with the gold (III) substance gold chloride (AuCl3). Tests conducted according to standard guidelines and are generally well-designed and well-reported. Measured test concentrations are slightly outside 80% of nominal.
Qualifier:
according to guideline
Guideline:
other: The growth inhibition assay was performed according to the methodology of Kershaw et al. (2005), Kim et al. (2011, 2012), and Li et al. (2009).
Principles of method if other than guideline:
Study performed according to the methodology of Kershaw et al. (2005), Kim et al. (2011, 2012), and Li et al. (2009).
GLP compliance:
no
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
A stock solution of 1000 mg Au(III)/L was prepared in Luria-Bertrani (LB) liquid medium and the stock solutions was diluted with LB liquid medium to obtain the treatment solutions.
Test organisms (species):
other: Escherichia coli and Bacillus subtilis
Details on inoculum:
- Test organisms: Gram-negative Escherichia coli (KACC10495) and gram- positive Bacillus subtilis (KACC10111)
- Source: Korean Agricultureal Culture Collection (KACC, Seoul, Korea)
- Culturing: Bacteria were cultured in LB liquid medium (2.5%) at 37 C, 135 rpm, and dark conditions overnight.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
12 h
Test temperature:
37 C
Nominal and measured concentrations:
Nominal concentrations of 0, 1, 5, 10, 15 and 20 mg Au(III)/L.
Maximum concentration of chloride used as gold salt was 3.6 mg Cl/L.
Details on test conditions:
- Medium: LB liquid medium
- Starting optical density: 1.1-1.2 at 600 nm
- Test vessel: 24-well plates (well-diameter 17 mm, height 17 mm) containing 2.5 mL test solution per well
- Initial cell density: 25 uL (1% v/v) of pre-cultured cell suspension was added to each well.
- Agitation: 135 rpm
Duration:
12 h
Dose descriptor:
EC50
Effect conc.:
31.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
tetrachloroauric acid
Basis for effect:
growth inhibition
Remarks:
(yield)
Remarks on result:
other: B. subtilis; effect concentrations reported based on Au(III), converted to tetrachloroauric acid, based on molcular weight.
Duration:
12 h
Dose descriptor:
EC50
Effect conc.:
24.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
tetrachloroauric acid
Basis for effect:
growth inhibition
Remarks:
(yield)
Remarks on result:
other: E. coli; effect concentrations reported based on Au(III), converted to tetrachloroauric acid, based on molcular weight.
Duration:
12 h
Dose descriptor:
NOEC
Effect conc.:
17.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
tetrachloroauric acid
Basis for effect:
growth inhibition
Remarks:
(yield)
Remarks on result:
other: B. subtilis; effect concentrations reported based on Au(III), converted to tetrachloroauric acid, based on molcular weight.
Duration:
12 h
Dose descriptor:
NOEC
Effect conc.:
25.88 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
tetrachloroauric acid
Basis for effect:
growth inhibition
Remarks:
(yield)
Remarks on result:
other: E.coli; effect concentrations reported based on Au(III), converted to tetrachloroauric acid, based on molcular weight.
Details on results:
The concentrations of Au(III) after the exposure duration were measured as 25.29 +/- 0.97 mg/L at 12 h for 20 mg/L dissolved in LB media. Measured concentrations for LB media (26.4%) were increased than nominal concentrations. Nominal concentrations were used for the calculation and reporting of results.

Effect concentrations converted to concentrations of tetrachloroauric acid (TCA) based on molecular weight:
Esherichia coli: 12-h EC50 14.2 mg Au(III)/L (24.5 mg TCA/L)
Bacillus subtilis: 12-h EC50 18.2 mg Au(III)/L (31.4 mg TCA/L)

Effect concentrations based on concentrations of Au(III):
12-h EC50 (B. subtilis, growth inhibition/yield): 18.2 mg/L (95% CL: 17.5-18.8 mg/L)
9-h EC50 (B. subtilis, growth inhibition/yield): 15.1 mg/L (95% CL: 14.3-15.9 mg/L)
6-h EC50 (B. subtilis, growth inhibition/yield): 10.5 mg/L (95% CL: 9.6-11.4 mg/L)
12-h NOEC (B. subtilis, growth inhibition/yield): 10 mg/L
12-h LOEC (B. subtilis, growth inhibition/yield): 15 mg/L
12-h EC50 (E. coli, growth inhibition/yield): 14.2 mg/L (95% CL: 13.4-15.0 mg/L)
9-h EC50 (E. coli, growth inhibition/yield): 12.1 mg/L (95% CL: 11.1-13.1 mg/L)
6-h EC50 (E. coli,, growth inhibition/yield): 11.4 mg/L (95% CL: 10.3-12.6 mg/L)
12-h NOEC (E. coli,, growth inhibition/yield): 15 mg/L
12-h LOEC (E. coli,, growth inhibition/yield): 20 mg/L
Reported statistics and error estimates:
The growth inhibition data of microorganisms were estimated as yield values, which were the biomass at the end of the test minus the starting biomass. The percentage inhibition yield at each concentration was calculated for the control, and expressed as growth yield. L(E)C50 values and corresponding 95% confidence limits for each species were estimated using the Trimmed-Spearman-Karber (TSK) Program.
Validity criteria fulfilled:
not specified
Conclusions:
Results reported in the study have been converted to concentrations of tetrachloroauric acid based on molecular weight. The 12-h EC50 was determined to be 24.5 mg/L tetrachloroauric acid (14.2 mg Au(III)/L) and 31.4 mg/L tetrachloroauric acid (18.2 mg Au(III)/L) for E. coli and B. subtilis, respectively.
Executive summary:

The acute toxicity of gold (III) chloride was assessed for Bacillus subtilis and Esherichia coli. Organisms were exposed to nominal concentrations of 0, 1, 5, 10, 15 and 20 mg Au(III)/L.for 12 hours in Luria-Bertani (LB) liquid medium. This study is reliable with restrictions as it was conducted according to a standard methodology and was well-reported, but no information on GLP compliance was reported. The 12-h EC50 was determined to be 24.5 mg/L tetrachloroauric acid (14.2 mg Au(III)/L) and 31.4 mg/L tetrachloroauric acid (18.2 mg Au(III)/L) for E. coli and B. subtilis, respectively. Effect concentrations were converted to concentrations of tetrachloroauric acid, based on molecular weight.

Description of key information

The 3-hour EC50 based on respiration rate was 27.9 mg/L. The NOEC was 2 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
27.9 mg/L
EC10 or NOEC for microorganisms:
2 mg/L

Additional information

In a GLP-compliant study following OECD guideline 209, a range-finding test was carried out using 3 concentrations of test item, the highest concentration was tested in triplicate (Brixham 2012). A definitive test was performed using 6 concentrations (5 replicates per test item) and a control. Results are reported as nominal concentrations. Although the validity criteria were not completely met this is not believed to have affected the study results. This study is therefore considered to be suitable for use for this endpoint. The 3-hour EC50 based on respiration rate was 27.9 mg/L. The NOEC was 2 mg/L.

Supporting data are available from a published guideline study for gold (III) chloride. Tests were conducted on two microorganism species with 5 test concentrations for 12 hours. Growth inhibition was determined based on biomass yield and results were reported as nominal concentrations (Nam et al. 2014). This study is considered to be suitable for use as a supporting study for this endpoint. Results reported in the study have been converted to concentrations of tetrachloroauric acid based on molecular weight. The 12-h EC50 was determined to be 24.5 mg/L tetrachloroauric acid (14.2 mg Au(III)/L) and 31.4 mg/L tetrachloroauric acid (18.2 mg Au(III)/L) for E. coli and B. subtilis, respectively.