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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, well designed and documented, no quantification of titanium concentration of test solutions
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Remarks:
Raw data was documented and balances as well as pipettes were used in the style of GLP. The quality assurance unit was not present and did not check the report; the report was not stored in an archiv.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Direct addition of test material into test vessel: no
- Sonication of stock dispersion: stock suspensions were prepared by dispersing the TiO2 material in deionized water for 15 min by using an ultrasound
probe (200 W, pulsed ultrasound: 0.2 s pulse and 0.8 s pause at 100% power, Sonoplus 200 W, BANDELIN Electronic GmbH & Co. KG). The preparation of the TiO2 stock suspensions (1 g/L) occurred directly before they were applied to the specific treatment groups.
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Test medium: synthtic sewage feed and activated sewage sludge according to OECD 209
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: Activated sludge was derived freshly from a domestic municipal waste water treatment plant [WWTP] (Bensheim, Germany or Rossdorf, Germany).
- Method of cultivation: The collected sludge was held in the lab for 2-3 days and was fed daily.
- Preparation of inoculum for exposure: After a settling period of 5-15 min coarse particles were removed from the sludge by decanting the upper water layer.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
no data
pH:
7.4 - 8.1
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
nominal concentrations: 10, 100, and 1000 mg n-TiO2/L
Details on test conditions:
TEST SYSTEM
- Test vessel: unspecified mixing vesel
- Type: open
- Aeration: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Weight of dry solids per volume of reaction mixture per unit of time:3 g dry weight/L
- Nutrients provided for bacteria: artificial sewage sludge according to OECD 209
- Nitrification inhibitor used (delete if not applicable): none
- In general, each replicate contained activated sludge (3 g dry weight per liter), tap water and, depending on the concentration a mixture of different ratios of deionized water and TiO2 stock suspension (TiO2 control).
- Only deionized water was applied to the controls.

OTHER TEST CONDITIONS
- Light intensity: laboratory light (according to OECD 209)
Reference substance (positive control):
yes
Remarks:
3,5- dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Hombikat UV 100; PC 105
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 101 and NM 102
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
EC50 value of the positive control (in mg n-TiO2/L):
- In test with NM-101: 3.56
- In test with NM-102: 4.34
Reported statistics and error estimates:
Data was statistically analyzed with ToxRat® Professional (version 2.10, ToxRat solutions GmbH). Significant differences between the treatment groups and the controls were determined using student-t test for homogeneous variances (two sided, *P<0.05).
Validity criteria fulfilled:
yes
Remarks:
exept for the NM-102 test, oxygen consumption of controls only 18.5 mg O2/ h*g dry weight instead of 20 mg O2/h* g dry weight
Conclusions:
In a 3 h activated sludge respiration inhibition test according to OECD 209, dispersions of the nano-sized, anatase TiO2 materials Hombikat UV 100 (7-10 nm) and PC 105 (15-25 nm) did not inhibit the respiration rate of the microorganisms of activated sludge, resulting in a 3 h-NOEC of ≥ 1000 mg/L (nominal, highest test concentration). The test with PC 105 is not valid, as validity criteria were not met.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 8-10, 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, well designed and documented, no quantification of titanium concentration of test solutions
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
In the reference test, the respiration rates at two treatment levels were assessed between 8.84 mg/L and 9.91 mg/L, which is outside the range of 6.5 mg O2/L to 2.5 mg O2/L that is recommended in the guideline. This was done due to low respiration rates
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not specified
Vehicle:
no
Details on test solutions:
- Direct addition of test material into test vessel: yes, The test solutions were prepared without using a stock solution. For each test item concentration level the test item was weighed into a separate weighing boat and transferred to the temperature-adapted test medium. Afterwards 200 mL of the microbial inoculum were added to each test vessel (controls and test item concentration levels). Immediately after preparation the test solutions were aerated.
- Sonication of dispersions: no
- Solvents: no
- Filtration or other separation of test material form test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersion: no
- Test medium: synthetic sewage feed and activated sewage sludge
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- microbiol inoculum
- Source: sewage treatment plant in Frankfurt/Main Niederrad, recieving predominantly domestic sewage

CULTURE CONDITIONS
- Date of sampling/ date of arrival at ECT: September 08, 2009
- Washing medium for activated sludge: Reconstituted water (OECD 1992)
- Frequency of washing: three times; between washings the activated sludge was centrifuged for 2 minutes at 1000 rpm
- Storage time before use: 2 days
- Storage temperature: 20 ± 2 °C
- During storage 50 mL synthetic sewage feed were added per litre activated sludge and per day. The activated sludge was stirred and aerated during storage.


SYNTHETIC SEWAGE FEED
-The synthetic sewage feed consisted of the following amounts of substances in 1 litre of deionised water (in g/L):
Peptone 16.0
Meat extract 11.0
Urea 3.0
NaCl 0.7
CaCl2 * 2 H2O 0.4
MgSO4 * 7 H2O 0.2
K2HPO4 2.8
The required amount of synthetic sewage feed was prepared not longer than 1 week before it was used.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.7 - 21.0
pH:
6.04
Nominal and measured concentrations:
nominal: control; 10, 32, 100, 320, and 1000 mg test item/L
Details on test conditions:
EXPOSURE CONDITIONS
- Storage time of the activated sludge before use: 2 days
- Test vessels: 1000 mL glass beakers without cover
- Amount of test mixture per test vessel: 500 mL
- Volume of microbial inoculum per test mixture: 200 mL
- Volume of synthetic sewage feed per test mixture: 16 mL
- Mixed liquor suspended solids level in the microbial inoculum: 4.23 g/L
- Mixed liquor suspended solids level in the final test mixtures: 1.69 g/L
- Number of test concentrations: 5 plus control
- Number of replicates per test item concentration: 2
- Number of replicates in the control: 2
- Aeration of test vessels: 50 to 60 litre of air per hour (ambient air, oil-free air-compressor)
Contact period: 3 hours
Temperature: 20.7 to 21.0°C (n = 39)



RECONSTITUED WATER
- Reconstituted water according to OECD Guideline No. 203 (Fish, Acute Toxicity Test) was used to wash and to maintain the activated sludge. The reconstituted water contained salts at the following final concentrations:
A) CaCl2 * 2 H2O 294.0 mg/L
B) MgSO4 * 7 H2O 123.25 mg/L
C) NaHCO3 64.75 mg/L
D) KCl 5.75 mg/L
After aeration for several hours the quality parameters of the reconstituted water were determined:
pH: 7.8
Hardness [mg/L CaCO3]: 253.6*
O2 concentration [mg/L] 7.9
O2 concentration [%] 94.7
Temperature [°C]: 23.8
Conductivity [µS/cm] 631
* Total hardness in mg/L CaCO3 was calculated by multiplication of the hardness-value in °dH by
17.86.
The required amount of reconstituted water was used within 4 weeks after preparation. During storage the reconstituted water was aerated.


PREPARATION OF TEST SOLUTIONS
- The test solutions were prepared without using a stock solution.
- The dilution medium (deionised water) was adapted to test temperature. For each test vessel (controls and test item concentration levels), 284 mL of the dilution medium were combined with 16 mL of the synthetic sewage feed. For each test item concentration level the test item was weighed into a separate weighing boat and transferred to the temperature-adapted test medium. Afterwards 200 mL of the microbial inoculum were added to each test vessel (controls and test item concentration levels). The total mixture volume for each of the test item concentrations and the controls was 500 mL per vessel. Table 1 describes the composition of the test mixtures and the controls. Immediately after preparation the test solutions were aerated.
- The test vessels were prepared at intervals of 15 minutes, starting with the first control, followed by the test item concentration levels. At the end of the test series the second control was prepared.
- At the end of the contact time (test period), the content of the first test vessel (control 1) was poured into the measuring apparatus (consisting of oxygen-meter, flat bottom flask and stirring device) and the respiration rate was determined at two-second intervals over a period of up to 10 minutes. All other test vessels were measured according to this procedure in the same order which was established for the preparation of the test vessels.
- The used microbial inoculum had a mixed liquor suspended solids level omixed liquor suspended solids level in the test solutions was 1.69 g/L.
- The vessels used had been sterilised in a drying oven for three hours at 150 °C.


METHOD FOR DETERMINING THE INHIBITION OF RESPIRATION
- An oximeter OXI 530 (WTW GmbH, D-82362 Weilheim) was used to determine the oxygen content of the test solutions. After three hours of incubation (contact time), the contents of the test vessels were poured into flat-bottom flasks. The oxygen electrode was fitted into the flat-bottom flasks which contained a stirrer bar. The flasks were placed onto a magnetic stirrer. The oximeter was connected to an automated data recording device. The oxygen measurements were taken at intervals of two seconds for up to 10 minutes at the end of the contact period for the content of each test vessel.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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:
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
Duration:
3 h
Dose descriptor:
LOEC
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:
Since the nominal test item concentrations of 10, 32, 100, 320, and 1000 mg test item/L showed no statistically significant inhibitory effect on the respiration rate of the test system, no ECx-values were determined.

Thus, the results of this study demonstrate that the test item did not inhibit respiration of activated sludge at a concentration of up to 1000 mg/L.

Toxic effect levels (EC20, EC50 and EC80) are therefore greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) is ≥ 1000 mg/L, and hence the Lowest Observed Effect Concentration (LOEC) is > 1000 mg/L.
Results with reference substance (positive control):
Control of the Test System (Reference Test) 3,5-dichlorophenol was used as the reference substance in a separate test on September 09, 2009. The same batch of sludge was used as in the definitive test. The effect of the reference item was determined to ensure that the test conditions were reliable. In the reference test 4 concentration levels (1.8, 5.9, 18.8, 60 mg/L) were tested. The reference test was performed according to ECT's SOP A 16.1. EC50 (3 h) = 5.0 mg/L The EC50 value of 5.0 mg/L is within the accepted range of 5 to 30 mg/L as required by the test guideline.
Reported statistics and error estimates:
The respiration rate was calculated from the oxygen consumption of the test solutions as mg O2/L • h (also expressed as mg O2/L • h) between 6.5 and 2.5 mg O2/L. The portion of the respiration curve over which the respiration rate was measured was linear.

STUDENT-t test for Homogeneous Variances with Bonferroni Adjustment was used to determine the No Observed Effect Concentration (NOEC) to compare each treatment with the control. Since in none of the treatments the respiration rate was inhibited statistically significant compared to the controls, the calculation of an ECx-value was not possible.

The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

Since the nominal test item concentrations of 10, 32, 100, 320, and 1000 mg test item/L showed

no statistically significant inhibitory effect on the respiration rate of the test system, no ECx-

values were determined.

Thus, the results of this study demonstrate that the test item did not inhibit respiration of

activated sludge at a concentration of up to 1000 mg/L.

Toxic effect levels (EC20, EC50 and EC80) are therefore greater than 1000 mg/L. The No

Observed Effect Concentration (NOEC) is ≥ 1000 mg/L, and hence the Lowest Observed

Effect Concentration (LOEC) is > 1000 mg/L.

Validity criteria fulfilled:
yes
Remarks:
All validity criteria were fulfilled as required by the study plan: The control respiration rates did not differ more than 15% compared to each other (CV ≤ 15%).; The EC50 (3 hours) of 3,5- dichlorophenol was in the accepted range of 5 to 30 mg/l
Conclusions:
In a 3 h activated sludge respiration inhibition test according to OECD 209, concentrations of a not further specified rutile TiO2 material of up to 1000 mg/L did not inhibit the respiration rate of the microorganisms of the activated sludge, resulting in a 3 h-NOEC of ≥ 1000 mg/L (nominal).
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, well designed and documented, no quantification of titanium concentration of test solutions
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
no
Remarks:
Raw data was documented and balances as well as pipettes were used in the style of GLP. The quality assurance unit was not present and did not check the report; the report was not stored in an archiv.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Direct addition of test material into test vessel: no
- Sonication of stock dispersion: stock suspensions were prepared by dispersing the TiO2 material in deionized water for 15 min by using an ultrasound
probe (200 W, pulsed ultrasound: 0.2 s pulse and 0.8 s pause at 100% power, Sonoplus 200 W, BANDELIN Electronic GmbH & Co. KG). The preparation of the TiO2 stock suspensions (1 g/L) occurred directly before they were applied to the specific treatment groups.
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Test medium: synthtic sewage feed and activated sewage sludge according to OECD 209
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: Activated sludge was derived freshly from a domestic municipal waste water treatment plant [WWTP] (Bensheim, Germany or Rossdorf, Germany).
- Method of cultivation: The collected sludge was held in the lab for 2-3 days and was fed daily.
- Preparation of inoculum for exposure: After a settling period of 5-15 min coarse particles were removed from the sludge by decanting the upper water layer.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
no data
pH:
7.4 - 8.1
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
nominal concentrations: 10, 100, and 1000 mg TiO2/L
Details on test conditions:
TEST SYSTEM
- Test vessel: unspecified mixing vesel
- Type: open
- Aeration: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Weight of dry solids per volume of reaction mixture per unit of time:3 g dry weight/L
- Nutrients provided for bacteria: artificial sewage sludge according to OECD 209
- Nitrification inhibitor used (delete if not applicable): none
- In general, each replicate contained activated sludge (3 g dry weight per liter), tap water and, depending on the concentration a mixture of different ratios of deionized water and TiO2 stock suspension (TiO2 control) or TiO2 stock suspension.
- Only deionized water was applied to the controls.

OTHER TEST CONDITIONS
- Light intensity: laboratory light (according to OECD 209)

Reference substance (positive control):
yes
Remarks:
3,5- dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tiona AT1
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Tiona AT1
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
EC50 value of the positive control (in mg n-TiO2/L): 4.19
Reported statistics and error estimates:
Data was statistically analyzed with ToxRat® Professional (version 2.10, ToxRat solutions GmbH). Significant differences between the treatment groups and the controls were determined using student-t test for homogeneous variances (two sided, *P<0.05).
Validity criteria fulfilled:
yes
Conclusions:
In a 3 h activated sludge respiration inhibition test according to OECD 209, concentrations of the pigement grade TiO2 material Tiona AT 1 of up to 1000 mg/L did not inhibit the respiration rate of the microorganisms of the activated sludge, resulting in a 3 h-NOEC of ≥ 1000 mg/L (nominal).

Description of key information

 Dispersions of nano- and microsized TiO2 do not inhibit microbial respiration in activated sewage sludge up to 1000 mg/L. 

Key value for chemical safety assessment

Additional information

Micro- and nanosized TiO2:

In one GLP-conform study and one study following the principles of GLP, activated sludge respiration inhibition tests according to OECD 209 were performed (i.e., Egeler and Goth, 2009; Wyrwoll et al. 2014). Results indicate that nano- and microsized TiO2 materials (including anatase and rutile) do not inhibit microbial respiration up to 1000 mg/L (unbounded 3 h NOEC: ≥ 1000 mg/L).

One supporting study with STP microorganisms demonstrates an absence of effects on the chemical oxygen demand of activated sludge microorganisms after long-term exposure (Wang et al. 2012). Exposures of microorganisms of activated sludge in lab-scale sequencing batch reactors to dispersion of the nano-sized TiO2 material Hombikat at concentrations between 0.5 - 2 mg/L (nominal) for 27 d did not significantly affect the chemical oxygen demand of the effluent in comparison to that of control batches. Mass balances of TiO2 in the whole system demonstrate that nominal and measured concentrations did not differ by more than 10%. Since the test duration exceeded typical STP sludge retention times in this study, results are considered as supporting data. Furthermore, one supporting study on the environmental fate of TiO2 nanoparticles in laboratory sewage treatment plants conducted according to OECD 303 A (Kuhlbusch et al. 2012; 5.6 Additional information on environmental fate and behaviour) showed no effect of TiO2 nanomaterials (P25, 21 nm) at concentrations between 1 - 10 mg/L (nominal) on DOC elimination and nitrification of the LSTP.

Several supporting studies with freshwater microorganisms are available. Tests investigated either i) effects on not relevant test organisms, such as the single species Vibrio fisheri (Kahru et al. 2007) or freshwater bacteria populations (Ozaki et al. 2016, Jomini et al. 2015), ii) not relevant endpoints, including endpoints that did not measure the function of microorganisms, but number of compromised cells, abundance, diversity and mortality (Battin et al. 2009, Jomini et al. 2015, Johnson et al. 2011). All these studies, except those of Battin et al. (2009) and Jomini et al (2015) indicate an absence of toxicity of nanosized TiO2 dispersions at concentration between 1 and 1000 mg/L to microorganisms. Battin et al. (2009) exposed free-living microorganism communities from natural waters to the nano-sized TiO2 materials P25 (~21 nm, rutile-anatase) and Hombikat UV 100 (< 10 nm, anatase), and found that exposure to P25 at 5.3 mg/L (nominal) for 24 h under dark conditions, caused an increase of compromised cells. Intracellular ROS generation was not significantly enhanced in these cells. Jomini et al. (2015) investigated the abundance and diversity of planktonic and sessile bacteria after exposure to dispersions of the nano-sized TiO2 material P25 (21 nm) in natural river water after 14 d of exposure under dark conditions. A 14 d exposure to 100 mg/L TiO2 caused an increase of planktonic bacteria, but a decrease of sessile bacteria compared to control abundance, however, significant differences were not observed at 1 and 10 mg/L. Further, the bacterial diversity was affected at 100 mg TiO2/L. However, Battin et al. (2009) and Jomini et al. (2015) did not assess endpoints relevant for the hazard assessment of nanosized TiO2 materials in STP.

In sum, all relevant and reliable studies indicate that micro- and nanosized TiO2 is not toxic to microorganisms in activated sewage sludge at concentrations up to 1000 mg/L.