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Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data obtained on Shk1 ( isolated from the activated sludge in an industrial WWTP), by the people who developped the method.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Bioluminescence inhibition of a genetically modified luminescent bacterium whose original strain was a Pseudomonas isolated from the activated sludge in an industrial WWTP.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
not specified
Details on test solutions:
Since some organic chemicals such as phenolic compounds have low water solubility, stock solutions were prepared by dissolving a desired amount of them in 10 ml acetone and then dissolved in 1000 ml distilled water. Assays solutions were made by making serial dilutions of the stock solutions to obtained the desired test concentrations
Test organisms (species):
other: Shk1 cells
Test type:
other: The Shk1 cells were supplied continuously by a CSTR (continuous-stirred tank reactor oprating at steady state. The Shk1 cells were alternatively mixed with buffer or test solutions at a ratio of 1:3 (volume)
Water media type:
freshwater
Total exposure duration:
5 min
Hardness:
No data
Test temperature:
22°C
pH:
7
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
No data
Details on test conditions:
The experimental setup consisted of a continuous-stirred tank reactor (CSTR) operated at steady state. The operating conditions of the CSTR in this study were a dilution rate of 0.52h-1, a pH of 7 and a temperature of 22°C. The CSTR was used to supply Shk 1 cells continuously. The Shk1 cells were alternately mixed with buffer (pH = 7) or test solutions at a 1:3 ratio (volume).
The bioluminescence of Shkl was measured after exposing Shkl cells to buffer or test solutions for 5 min.
Reference substance (positive control):
not specified
Duration:
5 min
Dose descriptor:
EC50
Effect conc.:
583 mg/L
Details on results:
- Experimental results:
Note that a compound's toxicity increases proportionally with the reciprocal of its ECso value (l/EC50).
In this study, authors obtained the EC50 values of the solubility-limited compounds from the extrapolation of a regression line obtained from the concentration-response data gathered up to the solubility limit. The extrapolation will add some additional uncertainties to the EC50 values of solubility-limited compounds, but because of their small number, their impact on the subsequent QSAR analysis is negligible. Since the EC50 and Kow, values span several orders of magnitude, they are expressed in logarithmic quantities.

- QSAR analysis:
The log(Kow)-dependent QSAR models have the following general form:
log(l /EC50, mM-1 ) = a log(Kow) + b (2)
A linear regression of log(l/EC50, mM-1) with log(Kow) yields a straight line, with the slope and intercept corresponding to the constants a and b in Eq. (2), respectively. After obtaining the regression line, the residuals were examined for possible outliers. In order to identify outliers, the semi-studentized residuals were formed according to the following equation:
e*=(e/MSE*10e1/2)

where e is the residual, e* is the semi-studentized residual, and MSE is the mean error sum of squares obtained in the regression.
Compounds that had a semistudentized residual of four or higher were identified as outliers and were removed from the group (see later discussion). A new regression equation was then obtained and residuals checked again to ensure the absence of outliers. Analysis of variance (ANOVA) was conducted to examine the statistical significance of the overall model. The Student-t test was performed to test the significance of the intercept and the slope of the regression line. The null hypotheses tested were (I) Ho: slope= 0 and (2) Ho: intercept = 0. The test on the slope examines whether the slope of the regression line is significantly different from 0. A slope that is significantly different from 0 indicates the existence of correlation between the two variables (in this case, log(l /ECso. mM-t) and log(K0w)). The test on the intercept examines whether the intercept of the regression line is significantly different from 0. If the intercept is not significantly different from 0, a linear regression through the origin should instead be performed.
For the groups of compounds, the slope and intercept of the regression line, number of compounds (n) used to generate the regression equation, outlier(s) removed, correlation coefficients (r), F-ratios and p values associated with ANOVA.

The correlation coefficients (r) range from 0.69 to 0.99. These r values are comparable to those obtained in QSAR analyses based on other organisms.
Validity criteria fulfilled:
not applicable
Executive summary:

The toxicity was measured using the Shk1 assay and EC50 values were obtained on 98 organic chemicals. EC50 (Shk1 bioluminescence inhibition) -5 min was 583 mg/l for guaiacol. It was not considered harmful for this mixed inoculum.

Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline, but test well described on a mixed inoculum. Netherless the test substance purity is not known.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Methanogenic activity inhibition test: evaluate the methanogenic toxicity to unacclimated methanogenic sludge.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Granular sludge (1 g of VSS) was transferred to the serum vials containing 100 mL of the basal medium and acetate was added from concentrated stock solution to obtain a final concentration of 2 g COD/L (readjusted by addition of acetate). The liquid was flushed with nitrogen gas and the flasks were sealed with a rubber septum and a screw cap and placed in reciprocating shaker at 30°C. Gaiacol was added to each flask to provide toxic concentrations to be investigated.
- Controls: yes
Test organisms (species):
anaerobic sludge
Details on inoculum:
- Preparation of inoculum for exposure: Elutriated sludge from a full scale UASB reactor treating distillery wastewater was used as inoculum.
The granular sludge was stored at 4°C and pre-acclimated by incubation at 30°C, in the presence of VFA. The sludge used was not previously acclimated to aromatic compounds.
Other parameters: no data
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
2 d
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 0.315 L glass serum flasks
Other parameters: no data

TEST MEDIUM / WATER PARAMETERS
- source preparation of dilution water: Basal medium consisting (in mg/L): NaHCO3 (400), NH4Cl (280), CaCl2 2H2O (10), K2HPO4 (250), MgSO4 7H2O (100), yeast extract (100). Per liter of medium, 1 ml of a trace element solution according to Zenhder et al (1980) was added.
- Total organic carbon: 2 g COD/L
Other: no data

OTHER TEST CONDITIONS: Specific methanogenic activity measurement was performed in 0.315 L glass serum flasks. The required amount of inhibitory compound was added to each flask to provide the toxic concentration to be investigated.
No toxicant was added to the controls. After flushing the headspace with nitrogen gas, the flasks were again incubated for 1 hour prior to the determination of the methane production rate.
The methane composition in the headspace content of each serum flasks was determined periodically during the 4 to 5 hour period that followed by gas chromatography.
The compound concentration that caused 50% and 80% inhibition of the methanogenic activity was determined by graphic method.

TEST CONCENTRATIONS: the toxicant concentrations were chosen as to cause an inhibition of the acetoclastic activity ranging from 0 to 100%.
Reference substance (positive control):
no
Duration:
2 d
Dose descriptor:
IC50
Effect conc.:
1 166 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: methanogenic activity
Duration:
2 d
Dose descriptor:
other: IC80
Effect conc.:
1 856 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: methanogenic activity
Details on results:
Authors' conclusion: Guaiacol was considered to have a toxic effect on the methanogenic activity of this unacclimated methanogenic sludge (IC50 < 7000 mg/L).
Reported statistics: the specific acetoclastic activity was calculated from the slope of the methane production versus time curve and the quantity of VSS (volatile suspended solid).
Validity criteria fulfilled:
not applicable
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was not a standardised method, but used a mixed inoculum. No information was available about the test substance.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Evaluate the methanogenic toxicity to anaerobic sludge by measuring their effect on the rate of methanogenesis from acetate.
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: granular sludge was added, to a final concentration of 0.5 volatile suspended solids (VSS) g/L, to 150-mL glass flasks containing 50 mL of cultivation medium supplemented with 30 mM acetate.
After purging air from the medium with nitrogen, the flasks were sealed with rubber stoppers and incubated anaerobically at 29+/-1°C under stirring. On the following day, the incubation medium was analysed for acetate and its concentration in medium was replenished to 30 mM.
The test substance was added to assay flasks at desired concentrations, missing control flasks free of tested substances. After flushing the flasks with nitrogen, the incubation was resumed. Following two days of incubation, the measurement and replenishment of acetate were repeated as described above. After a 1-h reincubation, the headspaces of all the assay flasks were analysed for methane produced during this time period.
Test organisms (species):
anaerobic sludge
Details on inoculum:
- Preparation of inoculum for exposure: The methanogenic microbial community under study represented granular sludge taken from an industrial UASB reactor of the wastewater treatment plant of a pulp-and-paper mill. Sludge was stored at 4°C, and prior to experiments, it was reactivated by incubating at 29°C in the presence of acetate.
Test type:
not specified
Water media type:
freshwater
Total exposure duration:
2 d
pH:
The final pH of the cultivation medium was 7.2-7.4
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 150 ml glass flask
- No. of vessels per concentration (replicates): no data
- No. of vessels per control (replicates): no data

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: contained (g/L): NH4Cl, 0.28; K2HPO4, 0.25; MgSO4.7H2O, 0.01; CaCl2.2H2O, 0.01; and 10 ml/L trace element solution.

OTHER TEST CONDITIONS
- Adjustment of pH: The final pH of the cultivation medium was 7.2-7.4.

EFFECT PARAMETERS MEASURED: inhibitory concentration (mg/L) at 20, 50 and 80%. It corresponds to methanogenic activity.
Toxicity of substances to methanogens was assessed by measuring their effect on the rate of methanogenesis from acetate. To this end, the methane production assay was continued for two days at 4 to 6-hours intervals. The maximal specific rate of acetoclastic methanogenesis was determined from the slope of the time curve of methane production. Inhibited methanogenic activity was expressed in percentage of the methane production rate in the control flasks.

TEST CONCENTRATIONS: NO DATA
Reference substance (positive control):
yes
Duration:
2 d
Dose descriptor:
other: IC20
Effect conc.:
806.91 mg/L
Conc. based on:
test mat.
Basis for effect:
other: methanogenic activity
Details on results:
Authors' conclusion: toxicity studies of some aromatic and cyclic compounds, among them gaiacol, as the probable product of biodegradation of certain surfactants, showed that methanogenesis in the microbial community under study are rather tolerant to high concentrations of these compounds.
Validity criteria fulfilled:
not applicable
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test well described but no information was available about the test substance.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Behavioural toxicity test: movement toward or away from a toxicant source
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: prepare a proteose peptone yeast extract (PPYE) medium by adding dextrose (0.5 g), proteose peptone (2 g), and finally yeast extract (2 g) to 400 ml of distilled water, stirring until the ingredients are dissolved. Autoclave in batch volumes as required below.
Test organisms (species):
Tetrahymena sp.
Details on inoculum:
Details on inoculum:
- Preparation of inoculum for exposure: 48 hours before the food deprivation pretreatment, transfer, using sterile technique, 1 ml of stock T. thermphila culture (ATCC 30382 Strain B-18684 [1975] or ATCC 30383 Strain B-18686 [1975], American Typa Culture Collection, Manasas, MD, USA) to 10 ml of sterile PPYE. After 24 h, transfer, using sterile technique, the 10 ml culture to 50 ml of sterille PPYE in a 250 ml Erlenmeyer flask. Then the corks for T-mazes should be soaked in commercially available spring water. After an additionnal 24h, centrifuge the 50 ml of PPYE culture in four 15 ml centrifuge tubes at 1200 rpm (300 g) for 3 min. With a Pasteur pipette, remove the supernatant, ensuring that the pellet of cells is not disturbed. Resuspended the 4 pellets of cells into one 15 ml centrifuge tube and then top up to 15 ml with spring water. Repeat the centrifugation 2 times, removing supernatant between steps and resuspending the pellet each time with approximatively 15 ml of spring water.
- Pretreatment: 18-h period of food deprivation in control water was applied before the test.
- Initial biomass concentration: 400 000 cells/mL (+/-10%)
Test type:
other: static, non renewal
Water media type:
freshwater
Total exposure duration:
20 min
Hardness:
measured at test initiation
Test temperature:
20°C +/-2°C, and measured at test initiation
pH:
measured at test initiation
Dissolved oxygen:
measured at test initiation
Nominal and measured concentrations:
0.1, 0.19, 0.38, 0.75 and 1.5 g/L.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: T-maze apparatus with glass T-mazes of test volume 5 ml
- No. of organisms per vessel: 10 to 20 µl of cells
- No. of vessels per concentration: 3
- No. of vessels per control: 1
- No. of vessels per vehicle control: 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: commercially available spring water
- Conductivity: measured at test initiation

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: no data
- Light intensity: Ambient laboratory light in the range 100-500 lux (diffuse, non-directional lighting)

EFFECT PARAMETERS MEASURED: Cell motility. LOEC based on calculation of Itox (number of cells migrating into arms of T-maze).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0, 6.25, 12.5, 25, 50 and 100%
- Results used to determine the conditions for the definitive study:
For each of 5 test concentrations in a dilution series, initially 100, 50, 25, 12.5, and 6.25%, perform the following motility test. Place approximately 10 to 20 µl of test cells in the bottom of each of five wells of a nine-well spot depression slide (Corning 7220, Thomas Scientific, Swedesboro, NJ, USA). Add the five test solutions in a 50:1 ratio (at the very least). Immediately, note the degree of cell motility as follows: acceptable motility - more than one half of the cells are mobile; or unacceptable motility - at least one half of the cells are not motile. After 5 min, assess cell motility again. The highest concentration in which motility is acceptable will become the highest concentration for the definitive 20-min test.
Reference substance (positive control):
yes
Remarks:
Sodium chloride at following concentrations: 625, 1250, 2500, 5000 and 10000 mg/L
Duration:
20 min
Dose descriptor:
LOEC
Effect conc.:
380 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: motility
Details on results:
Test organisms moved away from this reference toxicant. A reasonably good concentration-response relationship was found, with an LOEC of 380 mg/L (adjusted to pH 7.5) as the final endpoint and a mean Itox value at the LOEC of 0.24 (n=3).
Results with reference substance (positive control):
Results with reference substance (positive control): NaCl
Test organisms consistently moved toward this reference toxicant (attraction).
The LOEC was calculated as 2500 mg/L with mean Itox value at the LOEC of 0.66 (n=3).

- Other: Control treatments: An ANOVA was applied to the complete control data set and indicated that T. thermophila showed no preference for either arm of the T-maze. The mean Itox value was 0.50 with an SD of +/-0.062 (n=33).
Validity criteria fulfilled:
yes
Remarks:
The test was invalid if mean control Itox value is outside the range 0.43-0.57 and total cell counts (control and test arms added) for each replicate were =< 200 cells.
Conclusions:
Tetrahymena thermophila moved away from (ie., exhibited an avoidance reaction to) guaiacol. A reasonably good concentration-response relationship was found with a 20min-LOEC of 380 mg/l (adjusted to pH 7.5) as the final endpoint and a mean Itox value at the LOEC of 0.24 (n=3). The authors consider this value as much less sensitive than literature value for fish survival.
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: No sufficient information was available about the test condition (concentrations tested, control group, number of cells at the end of the study). Moreover, the tested species is of low relavance regarding STP microorganisms toxicity assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Effect upon the growth rate of yeast cells.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: the test substance was added in increasing amount to the medium, prior to the start of experiment. No details on preparation of test solutions.
Controls: a blank was carried alone with every experiment as control.
Test organisms (species):
Saccharomyces cerevisiae
Details on inoculum:
- Preparation of inoculum for exposure: the yeast cell suspension was prepared in a Wickerman medium, consisting of malt extract, yeast extract, peptone, (NH4)2SO4, MgSO4.H2O, glucose and H2O, adjusted with H3PO4 to pH 5.5. H2O was distilled from a glass apparatus. Fresh baker's yeast was obtained daily from a local distributor (Mautner-Markhof, Vienna). The viability of the cells was checked by setting up the growth curve in dependence of time.
- Initial biomass concentration: Cell density=10e8/ml
Test type:
static
Water media type:
freshwater
Total exposure duration:
210 min
Post exposure observation period:
no
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: erlenemyer flask
- No. of vessels per concentration: every experiment was repeated several times under identical conditions (n= 6+/-3)
- No. of vessels per control: 1 (blank)

TEST MEDIUM / WATER PARAMETERS: no data

OTHER TEST CONDITIONS: Viable yeast (150 mg) was suspended in the medium (10 mL) in Erlenmeyer flasks sealed with cotton plugs and shaken in a water bath at 30+/-1°C. Cell density was adjusted to approx. 10E+08/mL by appropriate dilution.
The test compound was added in increasing amount to the medium prior to the start of the experiment. A blank was carried along with every experiment as control.
The cell number was counted at 30, 90, 150 and 210 minutes of incubation time.

EFFECT PARAMETERS MEASURED: growth rate.
Two cell counting method were used : a manual method, where counting was performed in a blood cell counting chamber under microscope at 200-fold enlargement, and an automatized method, where a Coulter counter was used. The IC50 was estimated by graphic method.

TEST CONCENTRATIONS: no data
Reference substance (positive control):
no
Duration:
210 min
Dose descriptor:
IC50
Effect conc.:
227 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate
Reported statistics and error estimates:
log probit method

Description of key information

Key value for chemical safety assessment

Additional information

No key study has been selected regarding the endpoint of toxicity to microorganisms. However, a weight of evidence approach can be applied to assess the toxicity to STP microorganisms. Considering first the studies conducted on a mixed inoculum, the following results have been found :

- on Shk1 (genetically modified Pseudomonas extracted from activated sludge of an industrial WWTP) EC50 (bioluminescence inhibition) - 5 min=583 mg/l (Ren & Frymier, 2002)

- on anaerobic activated sludge (methanogenic activity inhibition): EC50 - 48H = 1166 mg/l (Sierra-Alvarez & Lettinga, 1989) and EC20 -48H = 806.9 mg/l (Shcherbakova et al., 1999).

Guaiacol has also been tested on individual bacterial species, likeTetrahymena thermophila (endpoint: motility): LOEC-20 min = 380 mg/l (Gilron et al., 1999).

Three other studies have been considered of low relevance regarding the assessment of STP microorganisms, they nevertheless led to similar results:

On Saccharomyces cerevisiae (growth rate): EC50 -210 min = 227 mg/l (Koch, 1992).

On Vibrio fischerii (bioluminescence inhibition) EC50-30 min = 92.03 mg/l (Nalecz-Jawecki et al., 2000)

On Escherischia coli (cell growth): EC50 -24H = 600 mg/l (Zaldivar et al., 2000)

All these results led to the conclusion that guaiacol is not harmful for microorganisms and that conclusion can be extrapolated to the STP microorganisms as well.