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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline study. However, study well documented, meets generally accepted scientific principles, useful as supporting information for hazard assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, the authors report the results of an experiment in which they grew three common species of stream algae as monocultures and together as polycultures in the biofilms of stream mesocosms that were exposed to nanoparticulate titanium dioxide (nTiO2) in a recirculating system.
GLP compliance:
not specified
Remarks:
not specified in the publication
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Water samples (50 ml) were collected from a well-mixed portion of the midchannel of each flume using a sterile syringe placed through a sampling septa.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Direct addition of test material into test vessel (dispersion): no
- Sonication of dispersions: Stock dispersions were produced by adding 1000 mg of n-TiO2 to 1.0 L water (E-pure water). The stock solution was sonicated for 30 min, after which inoculations of 1,287 ml (0.1 mg/L) or 12,873 ml (1 mg/L) were released into the running water of the appropriate streams using a pipette.
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersion: no, recirculating water phase; However, TiO2 dispersion was only applied once at test start.
- Test medium: A sterile soil- (fresh)water medium (SWM, detailed information see below)
Test organisms (species):
other: Synedra ulna, Scenedesmus quadricauda, Stigeoclonium tenue
Details on test organisms:
TEST ORGANISM
- Source: Culture Collection of Algae at the University of Texas (http://www.sbs.utexas.edu/utex/)
- Method of cultivation: The populations of algae used for the present study was maintained in nonaxenic laboratory cultures for approximately three months prior to the start of the experiment.

ACCLIMATION
- Acclimation period: 3 month
Test type:
other: recirculating test system, TiO2 was applied once at test start, but test medium recirculated with a flow rate of <2-55 cm/s
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Hardness:
no data
Test temperature:
no data
pH:
6.81 - 7.45
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
nominal concentrations: 0, 0.1, and 1.0 mg n-TiO2/L
measured concentrations:
- test start: 0, 0.1, and 1.0 mg n-TiO2/L
- test end: 0.015 (= background), 0.015, and 0.026 mg n-TiO2/L

- graphical estimate: until 10 d after test start a TiO2 concentration of ~ 0.08-0.10 mg TiO2/L was detected in the water phase of the 1 mg/L (nominal) treatment group, whereas the TiO2 concentration in the 0.1 mg/L (nominal) treatment group directly declined to ~0.015 mg/L after 2 d.
Details on test conditions:
TEST SYSTEM
- Test vessel: artificial flume containing vertical angled PVC substrates
- Material, size, headspace, fill volume: flume: 0.5 m x 0.1 m x 0.1 m;
- velocity: < 2 to 55 cm/s
- Type of flow-through (e.g. peristaltic or proportional diluter): recirculating test system
- Initial cells density: 1.65 E5 cells
- No. of vessels per concentration (replicates): 5 (algae free, innoculated with soil extract medium only); 5 (single algae); 7 (polyculture)
- No. of vessels per control (replicates): 5

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: A sterile soil- (fresh)water medium (SWM) was used for culturing algae. SWM is made by steeping greenhouse potting siol for 24 h in UV sterilized E-pure water to leach micro. and macronutrients that are required for growth. The growth medium was prepared according to Watanabe (2005) and analysed.

TEST MEDIUM PARAMETERS
- Source/preparation of dilution water: natural freshwater
- Total organic carbon: 5283 µM C
- Metals: AL, As, B, Ba; Ce, Cu, Fe, I, Li, Mn, P, Rb, S, Si, Sr, Ti, Zn
- Chlorine: 103.3 mg/L
- Ca/mg ratio: 17.66 mg/L
- Conductivity: 372.0 µS
- Intervals of water quality measurement: During the test conductivity, pH value, and the natural organic matter were measured at test day 1, 4, 11, 22, and 31.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: not reported
- Light intensity and quality: Coralife T5 Aquarium light fixture containing two 9-watt, 10K daylight spectrum fluorescent lamps (with irradiance peaks at 550 and 610 nm, and in the upper UV spectrum at low intensities at 360–400 nm, intensities of >0.2mW/cm² were apparent).

Reference substance (positive control):
not specified
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
biomass
Reported statistics and error estimates:
The repeated measures mixed model analyses of variance (ANOVA) was used with an autoregressive covariance matrix to model the biomass of each biofilm as a function of the initial concentration of nTiO2, the day of the experiment, and the nTiO2 time interaction.
Any significant interaction was taken as evidence that nTiO2 alters the growth trajectory of algal biomass. General linear models were then used to assess how the final biofilm biomass and the final concentration of Ti in the biofilm varied as a function of the initial concentrations of nTiO2 and biofilm composition. All analyses were performed using SAS Version 9.1.3 (SAS Institute).

Validity criteria fulfilled:
not specified
Conclusions:
Algae biofilms consisting of monocultures of either Synedra ulna, Scenedesmus quadricauda, or Stigeoclonium tenue as well as biofilms consisting of polycultures of these three species were exposed to dispersions of the nanosized TiO2 material P25 (27 ± 4 nm) in a recirculating system for 32 d. No significant effects on biomass were observed in biofilms of monocultures and polycultures, resulting in 32 d-NOEC values of ≥ 1 mg n-TiO2/L (nominal). TiO2 concentrations in the water phase of the 0.1 and 1 mg/L treatment groups decreased either to background levels (0.015 mg/L) or 0.026 mg/L at the end of the test.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: study experiment is not guideline-followed and test concentrations were not measured
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, the authors report the results of an experiment in which they grew four common species of marine algae in Erlenmeyer glass flasks and exposed them to nanoparticle titanium dioxide (nTiO2).
GLP compliance:
not specified
Remarks:
not specified in the publication
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 dispersions: 10 mg of n-TiO2 was added to 1 mL of filtered (0.2 µm Millipore) natural seawater and sonicated for 60 s, vortexted briefly, and diluted to 10 mg/L.
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Appearance of dispersion: no information
- Renewal of test dispersion: no, static
- Test medium: filtered (0.2 m Milipore) natural seawater
Test organisms (species):
other: Thalassiosira pseudonana, Skeletonema marinoi, Dunaliella terlilecta, and Isachrysis galbana
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Provasoli-Guillard National Center for Culture of Marine Phytoplankton (Bigelow Laboratory for Ocean Schience, West Boothhay Harbor, Maine, USA)
- Method of cultivation: Standard medium made with filtered (0.2 µm), autoclaved natural seawater and cultivated under test conditions.

ACCLIMATION
- Acclimation period: not reported
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
no data
Test temperature:
15 °C
pH:
no data
Dissolved oxygen:
no data
Salinity:
34 ppt
Nominal and measured concentrations:
nominal concentration: 0, 0.01, 0.1, 0.5, ans 1 mg n-TiO2/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Material, fill volume: glass, 500 mL fill volume, 200 mL media volume
- Aeration: 5-7- days until log-phase growth
- Initial cells density: 1-2 E5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Cell density was counted every 24 h by using a hemacytometer

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural seawater
- Intervals of water quality measurement: every 24 h

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 14 h light/ 10 h dark
- Light intensity and quality: cool white fluorescent light (100 - 120 µmol m²/s)
- Salinity (for marine algae): 34 ppt
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
AEROXIDE TiO2 P25
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
for: S. marinol, T. pseudonana, D. tertiolecta and I. galbana
Details on results:
Exposure to nano-TiO2 after 96 h did also not affect the growth rate of S. marinol, T. pseudonana, D. tertiolecta and I. galbana. Resulting in a unbounded NOEC of ≥ 1 mg/L.
Reported statistics and error estimates:
Phytoplankton population growth rates for each replicate flask were estimated as the lope of log-transformed cell count data, obtained through least-square regression.
One-way ANOVA was used to test for an overall effect of nanoparticle toxicity on growth rates.
Validity criteria fulfilled:
not specified
Conclusions:
Dispersions of a nanosized TiO2 material from Evonik Degussa (15-20 nm) up to the highest test concentration of 1 mg/L did not significantly reduce the growth rate of four phytoplankton species, including diatoms, chlorophytes and prymnesiophytes in a non-standardized toxicity test. Reported 72 h NOEC-values were ≥ 1 mg n-TiO2/L (nominal) for S. marinol, T. pseudonana, D. tertiolecta, and I. galbana.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: no quantification of titanium dioxide of test dispersions; additional tests were performed in 24-well plates (shaking) and in cylindrical vials (25 ml, stirring bar)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Principles of method if other than guideline:
Tests were performed either in Erlenmeyer flasks, 24-well plates or in small glass vials. Erlenmeyer flasks and 24-well plates were continuously shaken and medium in cylindrical glass vials was stirred.
GLP compliance:
not specified
Remarks:
not specified in the publication
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 dispersion: TiO2 nanopowder was added to ultrapure water and homogenised by manual stirring. After obtaining the stock concentration of 1 g/L by adding of additional water the dispersion was sonicated for 15 minutes. The increase of temperature during preparation was reduced by an ice bath.
- Solvents: no
- Filtration or other separation of test material from test dispersion: no
- Test medium: OECD algae growth medium
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Korshikov
- Age of inoculum (at test initiation): three days
- Method of cultivation: cultured in laboratory

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
22 ± 1 °C
pH:
8.2
Dissolved oxygen:
not stated
Salinity:
not stated
Conductivity:
not stated
Nominal and measured concentrations:
Range of concentrations: 0, 2.1 to 50 mg n-TiO2/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flask
- Material, size, headspace, fill volume: glass, 100 mL
- Initial cells density: 8 x 10³ algal cells per mL
- Control end cells density: not stated
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6; control medium (without algae) was also subjected to sonication
- shaking: 125 rpm

Additional tests were performed in 24-well plates (2.5 ml/well, 125 rpm) and in cylindrical glass vials (25 ml). 24-well plates were shaken and the media in cylindrical glass vials was stirred with magnetic stirring bars.

GROWTH MEDIUM
- Standard medium used: OECD algae medium


OTHER TEST CONDITIONS
- Sterile test conditions: not stated
- Adjustment of pH: not stated
- Photoperiod: continuous illumination
- Light intensity and quality: 5000-6000 lux; fluorescent light (OSRAM Fluora 77)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence measurement with microplate reader. Excitation wavelength: 438 nm and emission wavelength: 685 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: < 3.2

REFERENCE SUBSTANCE AND ADDITONAL CONTROLS
- potassium dichromate tested in all test vessels: results show that the exposure system did not modify the response of potassium dichromate.
- TiO2 control (50 mg/L; TiO2 without algae): These controls were fluorometrically measured. No fluorescence from TiO2 nanoparticle dispersions observed in all test vessels.
Reference substance (positive control):
yes
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 101
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Erlenmeyer flasks
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 101
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Erlenmeyer flasks
Duration:
72 h
Dose descriptor:
other: EC25
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 101
Basis for effect:
growth rate
Remarks on result:
other: Erlenmeyer flasks
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 101
Basis for effect:
growth rate
Remarks on result:
other: Erlenmeyer flasks
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 104
Basis for effect:
growth rate
Remarks on result:
other: Erlenmeyer flasks
Duration:
72 h
Dose descriptor:
other: EC25
Effect conc.:
28.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 104
Basis for effect:
growth rate
Remarks on result:
other: Erlenmeyer flasks
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
NM 104
Basis for effect:
growth rate
Remarks on result:
other: Erlenmeyer flasks
Details on results:
AGGLOMERATION BEHAVIOUR

- Stock dispersion:
Hydrodynamic diameters (Z-average in intensity): 464 and 172 nm for NM 101 and NM 104, respectively
Zeta potentials: -27 mV and +23 mV for NM 101 and NM 104, respectively

- Test medium:
Hydrodynamic diameters (Z-average in intensity): 763 nm and 1224 nm for NM 101 and NM 104, respectively
Zeta potentials: -18 mV and 12 mV for NM 101 and NM 104, respectively

ADDITIONAL TESTS
- Growth inhibition by NM 101 and NM 104 was more pronounced when tested in 24-well plates and cylindrical vials compared to when algae were exposed to these test materials in Erlenmeyer flasks, resulting in 72 h-NOEC, EC25 and EC50 values of < 2.1, 0.2 and 8.5 mg/L, and < 2.1, 0.2 and 2.7 mg/L for NM 101 in 24-well plates and cylindrical vials, respectively. For NM 104 72 h-NOEC, EC25 and EC50 values accounted < 2.1, 10 and > 50 mg/L, and < 2.1, 7.9 and 39 mg/L for NM 104 in 24-well plates and cylindrical vials, respectively.

FLOW CYTOMETRY ANALYSIS
- Increase of complex cells (related to a direct interaction of particles and algae, either by adsorption or internalization) increased by 15 % in cylindrical vials compared to 1.2% for cells exposed in Erlenmeyer flasks at 2.1 mg/L NM 101.
- Affinity of NM 104 to algal cells was generally less pronounced compared to NM 101 particles.
Results with reference substance (positive control):
Comparable results observed from all test systems.
Reported statistics and error estimates:
Calculation of toxicity parameters (ECx) was determined using the logistic Hill model, whereas the 95% confidence interval was determined via a bootstrap simulation model. All analysis was performed with the REGTOX_software (v.7.0.5).
Validity criteria fulfilled:
yes
Remarks:
only stated in the text, no specification.
Conclusions:
Dispersions of the two titanium dioxide nanoparticles NM 101(< 10 nm) and NM 104 (20 nm) were tested in Erlenmeyer flasks in an algae toxicity test according to OECD 201 (2011). Determined EC10, EC25 and EC50 values for growth rate amounted to < 2.1, 2.1, 10 and > 50 mg/L for NM 101, and (not determined), 28.7, and > 50 mg/L for NM 104.

Additionally, algae were exposed to NM 101 and NM 104 in 24-well plates (continuous shaking) and cylindrical glass vials (stirring with magnetic bar). Results indicate that growth inhibition was more pronounced under these test conditions. However, results of these experiments are not considered for the hazard assessment of nano-TiO2, because it is expected i) that due to the low exposure volume, evaporation of the test medium in 24-well plates caused a significant increase of exposure concentration during the testing period, and ii) that pronounced growth inhibition in cylindrical vials was a consequence of using a stirring bar, which might have caused destruction of algae cells through grinding of the cells with agglomerated and precipitated TiO2 particles.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
January 11-26, 2007
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: GLP guideline study; meets national standard methods
Qualifier:
according to
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Version / remarks:
1998
Principles of method if other than guideline:
The method assesses the growth rate of cultures in solutions of test material in enriched seawater in comparison to the growth rate of cultures in enriched seawater alone. Growth rate was measured in terms of increase in cell number or in biomass.
GLP compliance:
no
Remarks:
Study performed under GLP, certificate not shown and GLP statement not signed
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- Direct addition of test material into test vessel (dispersion): yes, but supernatants were prepared by the direct addition of the required nominal weights or volumes to seawater followed by gentle stirring for approximately 20 hours followed by a settling period of approximately one hour. After this settling period, the middle phase of the preparation is siphoned, avoiding incorporation of undissolved particles, if present.
- Sonication of dispersions: no
- Solvents: no
- Filtration of other separation of test material from test dispersion: After the settling period of hour, the middle phase of the preparation is siphoned, avoiding incorporation of undissolved particles, if present.
- Apperance of dispersion: floating or settled particles at 1000 mg/L in sea water after 20 h of stirring (before separating the sedimented particles)
- Test medium: natural seawater (see below)
Test organisms (species):
Skeletonema costatum
Details on test organisms:
- Pre-cultures in the exponential growth phase were prepared from stock laboratory cultures by inoculating nutrient medium (ISO culture medium) to a cell density of approximately 2 x 10E3 to 10E4 cells per milli litre.
- The pre-cultures were incubated at approximately 20±2 °C under constant illumination for 3 d ±1 d, and
were used as the inoculum source for subsequent toxicity tests.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
18.8-19.5 °C
pH:
8.14-8.19
Salinity:
35 ppt (salinity of dilutent water at 0h)
Nominal and measured concentrations:
1000, 1800, 3200, 5600, 10000 mg/l
Details on test conditions:
TEST PROCEDURE
- 100 ml borosilicate glass conical flasks, to which 80 ml of test medium seawater was added.
- Each treatment was prepared in duplicate, and inoculated with cells from the precultures (in exponential growth phase) to give an initial cell density of approximately 10,000 cells per ml.
- controls containing only test medium were prepared with four replicates
- The initial inoculum was checked microscopically using a haemocytometer. Following inoculation, all flasks were loosely covered with aluminum foil caps, and mounted on an orbital shaker (approximately 18 mm orbit, 150 rpm) under cool white illumination as specified in the ISO guidelines.
- five concentrations and two replicates per concentration.

ILLUMINATION
- four 40W tubes mounted at a distance of approximately 40 cm directly above the test area.
- Light intensity: 7730-8580 lux. Light intensity values were measured daily during the test. Flasks were assigned positions on the shaker.

REFERENCE TEST
A reference test was conducted concurrently using 3,5 Dichlorophenol at 5.6, 3.2 and 1.8 mg.l-1 which were prepared from a main stock of 100 mg.l-1. The 100 mg.l-1 stock was stirred for a minimum of one hour, or until completely dissolved. ISO medium is prepared from natural seawater supplied by pump from Scapa Flow, Orkney.

TEST MEDIUM:
All seawater was UV sterilised and filtered to 0.2 µm. The filtered treated seawater was then enriched with nutrients and vitamins in accordance with ISO guidelines. The salinity of the enriched natural seawater at 0h was 30‰ ± 5‰. At pre 0h or 0h, the pH of the ISO culture medium was adjusted if required, by adding 1M HCl, or NaOH to give a pH of 8±0.2. If, at 0h the pH of the test material stock(s) was outwith the pH range of 8±0.2 then the pH was returned to within these limits by adjustment with either 1M HCl or NaOH as was appropriate. If the pH requires adjustment, a stirring period was required to ensure the pH remained constant.
Reference substance (positive control):
yes
Remarks:
reference toxicant 72h EC50: 2.76 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
in supernatant (siphoned after 1 h of sedimentation following 20 h of stirring)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5 600 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
in supernatant (siphoned after 1 h of sedimentation following 20 h of stirring)
Basis for effect:
growth rate
Remarks on result:
other:
Reported statistics and error estimates:
The raw data for each duplicate vessel and time period were averaged, to give values for each concentration of cell volume. Growth rate was calculated on the basis of these measurements. Daily intrinsic growth rate was calculated for each duplicate for each time period, using an exponential model.
The resulting values represent proportional reduction in growth rate. The EC50 for each time interval and the 72h EC90 and NOEC values were calculated using an appropriate statistical method from the ToxCalc Version 5 software.

72h EC90: >10000 mg/l

72h NOEC: 5600 mg/l

24h EC50: >10000 mg/l

48h EC50: >10000 mg/l

Validity criteria fulfilled:
yes
Remarks:
in accordance with the study plan and met all relevant validity criteria
Conclusions:
Exposure of the marine algae Skeletonema costatum to supernatants of a not further specified TiO2 material, prepared by stirring and siphoning of the middle phase after a sedimentation period of 1 h, in an algae toxicity test according to the ISO 10253 guideline resulted in 72 h-NOEC values of 5600 mg TiO2/L (nominal).
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
November 16 - 24, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Duplicate samples of approximately 20 mL were taken.
- Sample storage conditions before analysis: One sample per treatment was stored at -20 °C as retain sample and one sample was analyzed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Direct addition of test material into test vessel: yes; All work was performed under sterile conditions and the handling of the test item was done with maximum possible light avoidance. Each loading rate was prepared separately. Amounts of 20, 36, 64, 112 and 200 mg of the test item were added to 2 L growth medium under sterile conditions in a 2 L glass flask with a drain port near the bottom. The preparation was stirred at room temperature for 48 h at 300 rounds per minute.
- Sonication of dispersions: no
- Filtration or other separation of test material form test dispersion: To remove non-dissolved test item from the test medium, each test preparation was filtered using a 0.1 µm Pall Acrodisk 25 mm syringe filter without pre-filtration.
- Appearance of dispersion: While stirring sedimentation of the test item was observed. After filtration a clear liquid was present.
- renewal of test dispersion: no, static
- Test medium: AAP growth medium according to the OECD guideline 201
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Source: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut (Göttingen, Germany).
- Age of inoculum (at test initiation):
- Method of cultivation: The stock cultures are maintained fulfilling the criteria of the OECD 201 guideline.

ACCLIMATION
- Culturing media and conditions: Prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
22 ± 0.1 °C
pH:
The pH of the controls increased from 7.91 up to a range of 8.46 - 8.89 during the test.
In the test cultures the initial pH was 7.45 - 7.69 and was 8.39 - 8.87 at the end of the test.
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
nominal concentrations: 10, 18, 32, 56 and 100 mg TiO2/L
measured concentrations were below LOQ (0.8610) and max 1.5 µg TiO2/L; The titanium concentration in the solution after 48 h stirring and the filtration step did not depend on the loading rate. After 72 h of exposure all values for Ti were below the LOD or LOQ.
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks
- Type: closed (covered with air-permeable silicone-sponge caps)
- Material, size, heads pace, fill volume: glass, 250 mL
- Initial cells density: 10000 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes ( sterilised synthetic AAP medium according to OECD 201)

OTHER TEST CONDITIONS
- Light intensity and quality: 7251 - 7312 lux
- Sterile test conditions: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
in filtrate (0.1 µm filtered after 48 h of stirring)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
in filtrate (0.1 µm filtered after 48 h of stirring)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
filtrate, 0.1 µm filtered after 48 h of stirring
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
filtrate, 0.1 µm filtered after 48 h of stirring
Basis for effect:
biomass
Details on results:
For the measurement parameters growth rate and yield (biomass), the EC50, EC20 and EC10 were not calculated, since no concentration dependent effect was observed and the strongest inhibition was 6.2 % compared to controls for yield at a loading rate of 32.0 mg/L at test end.
Therefore, the EC50 values were set to be > 100 mg/L, the highest loading rate. The algal growth inhibition test revealed a NOEC for titanium dioxide of ≥ 100 mg/L.
Results with reference substance (positive control):
- EC50: The most recent nominal ErC50 value of 3.78 mg/L (95 % confidence limits 3.64 - 3.92 mg/L) is in good agreement with the results of an international ring test with ErC50 of 3.38 ± 1.30 mg/L.
Reported statistics and error estimates:
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on the nominal loading of the test medium. The mean value of the cell counts for each concentration plot was used for plotting growth curves. Calculation of the percent inhibition of growth rate [r] and yield [y] was performed according to the guideline OECD 201. The percent inhibition values were plotted as a function of the test item concentration. The test results were statistically analyzed to determine EC10 and EC50 values with 95 % confidence intervals using Probit-analysis [9] assuming log-normal distribution of the values by using the computer program ToxRat®. The NOEC and LOEC values were determined by the Williams Multiple Sequential t-test or the Dunnett’s multiple t-test. The computer program ToxRat Professional was used for statistical evaluations.

Table: Chemical analysis of test item, results [µg/L].

 

Ti concentration [µg/L]

 

Treatment [mg/L]

Control

10.0

18.0

32.0

56.0

100

LOD

LOQ

Test start

<LOQ (0.6151)

0.9095

<LOQ (0.7653)

0.9571

<LOQ (0.6009)

0.9116

0.2870

0.8610

Test end

<LOD

<LOD

<LOD

<LOQ (0.5445)

<LOD

<LOQ (0.5722)

0.4736

1.4209

 

TiO2* concentration [µg/L]

 

 

Treatment [mg/L]

Control

10.0

18.0

32.0

56.0

100

LOD

LOQ

Test start

<LOQ (1.026)

1.518

<LOQ (1.277)

1.597

<LOQ (1.003)

1.521

0.4789

1.437

Test end

<LOD

<LOD

<LOD

<LOQ (0.9086)

<LOD

<LOQ (0.9548)

0.7904

2.371

*The TiO2concentration was calculated based on a purity of the test item of 98.72% as stated in the certificate of analysis.

Validity criteria fulfilled:
yes
Conclusions:
Dissolved/dispersed fractions of a pigment grade TiO2 material, which were prepared by stirring the test material in test medium for 48 h and by subsequently filtering this test dispersion through a 0.1 µm filter, did not significantly affect the growth rate and biomass yield of Pseudokirchneriella subcapitata in an algae growth inhibition test according to OECD 201 after 72 h of exposure (72 h-NOEC ≥ 100 mg/L; 72 h-LOEC > 100 mg/L, nominal). Measured TiO2 concentrations in the test medium (10-100 mg TiO2/L, nominal) at test start ranged from 1.52-1.60 µg TiO2/L or were smaller than the LOQ (1.437 µg TiO2/L), and did not depend on the TiO2 loading rate.

Description of key information

Dispersions of microsized TiO2 are not toxic to aquatic algae and cyanobacteria up to ≥ 100 mg/L (NOEC) and 5,600 mg TiO2/L (NOEC) in fresh- and saltwater, respectively. Considering the low solubility of microsized TiO2, it is further concluded that microsized TiO2 is not toxic to freshwater algae up to its solubility limit. A low toxic potential of algal toxicity is observed for dispersed nanosized TiO2 with EC10 and EC50 values being > 2 mg/L and > 50 mg/L, respectively.

Key value for chemical safety assessment

Additional information

Microsized TiO2:

Dissolved/dispersed pigment grade TiO2 material, prepared by stirring for 48 h and filtering the dispersion through a 0.1 µm filter, did not significantly affect the growth rate of Pseudokirchneriella subcapitata in a test according to OECD 201 (72 h NOEC ≥ 100 mg/L, nominal). Measured TiO2 concentrations of the test media (10-100 mg TiO2/L, nominal) at test start were below the LOQ (1.44 µg TiO2/L) or ranged from 1.52-1.60 µg TiO2/L and did not increase with the TiO2 loading rate (Schlich et al. 2015). Thus, microsized TiO2 is not toxic to algae up to its solubility limit.

Microsized TiO2 was also not toxic to marine algae. Hudson et al. (2007) exposed Skeletonema costatum to the supernatant of TiO2 dispersions, prepared by stirring for 20 h and siphoning of the middle phase after a sedimentation period of 1 h, in a test according to ISO 10253. A 72 h NOEC value of 5600 mg TiO2/L (nominal) was derived. It is further concluded that microsized TiO2 is not toxic to marine algae up to its solubility limit.

In the study by Hudson et al. (2007), TiO2 particles at these unrealistically high concentrations may have adsorbed nutrients that are necessary for algae growth including phosphate and thereby caused algae growth limitation in the closed, artificial test system. Hence, effect values as derived by Nicolas et al (2015) may be due to an experimental artefact and considered overly conservative.

The algae studies were performed at concentrations several magnitudes above the solubility limit of microsized TiO2. Transformation/dissolution data of different microsized TiO2 materials indicate a low solubility in environmental media as dissolved Ti concentrations after 28 d were below the respective LOD/LOQ (< 0.11 / < 0.34 µg Ti/L). In sum, it is concluded that microsized TiO2 is not toxic to algae up to the solubility limit in fresh- and saltwater.

Nanosized TiO2:

Nicolas et al (2015) tested dispersions of the two titanium dioxide nanoparticles, i.e. NM 101 (< 10 nm) and NM 104 (20 nm), in Erlenmeyer flasks in an algae toxicity test according to OECD 201 (2011). The determined EC10 for inhibition of growth rate amount to 2.1 and 4.7 mg/L for NM 101 and NM 104, respectively, whereas the EC50 were > 50 mg/L indicating a low potential for toxicity to algae.

Nano-TiO2 may adsorb nutrients that are necessary for algae growth including phosphate. Several studies have shown sorption of phosphate to nano-TiO2, such as Luo et al. 2015 and Moharami et al. (2014). Hence, nanosized TiO2 may have adsorbed nutrients, such as phosphate, from the test medium and thereby caused algae growth limitation in the closed, artificial test system. Due to a bigger and open nutrient pool in natural open environments, it is not expected that nutrient adsorption by nanosized TiO2 at concentrations used by Nicolas et al (2015) would cause algal growth limitations. Hence, effect values as derived by Nicolas et al (2015) may be due to an experimental artefact and considered overly conservative.

One study on the long-term toxicity of nanosized TiO2 to freshwater polycultures further indicates that nano-TiO2 is not toxic to biofilms at 1 mg/L. In this study by Kulacki et al. (2012), algae biofilms consisting of monocultures of either Synedra ulna, Scenedesmus quadricauda, or Stigeoclonium tenue as well as biofilms consisting of polycultures of these three species were exposed to dispersions of the nanosized TiO2 material P25 (27 ± 4 nm) in a recirculating system for 32 days without any significant effects on biomass resulting in 32-d NOEC values of ≥ 1 mg n-TiO2/L (nominal). Dispersed TiO2 concentrations in the water phase of the 0.1 and 1 mg/L treatments decreased to background levels (0.015 mg/L) or to 0.026 mg/L at the end of the test, respectively.

Miller et al. (2010) also found that nanosized TiO2 was not toxic to marine phytoplanktonic algae up to 1 mg/L. Dispersions of a nanosized TiO2 material (15-20 nm) did not significantly reduce the growth rate of four marine phytoplanktonic species, including diatoms, chlorophytes and prymnesiophytes, in a non-standardized toxicity test. Reported 72 h NOEC-values were ≥ 1 mg n-TiO2/L (nominal).

In sum, two GLP-conform guideline tests of the toxicity of microsized TiO2 to aquatic algae and cyanobacteria resulted in an unbounded NOEC value of ≥ 100 mg/L TiO2/L for Pseudokirchnerella subcapitata in freshwater and a NOEC of 5,600 mg TiO2/L for Skeletonema costatum, in saltwater. Nicolas et al (2015) observed some toxicity of two n-TiO2 materials, however, the respective EC10s are well above long-term classification criteria. Studies with n-TiO2 and freshwater polycultures (Kulacki et al. 2012) or saltwater phytoplankton species (Miller et al. 2010) point to a low potential for toxicity to algae. In sum, it can be concluded that microsized TiO2 is not toxic to aquatic algae and cyanobacteria up to ≥ 100 mg/L (NOEC) and up to 5,600 mg TiO2/L (NOEC) in fresh- and saltwater, respectively. Also, a low potential for algal toxicity is observed for nanosized TiO2 with EC10 and EC50 values being > 2 mg/L and > 50 mg/L, respectively.

References:

Luo et al. 2015. The Effect of Phosphate and Sulfate on Arsenate Desorption From Nano-TiO2. J. Res. Sci. Tecnol. 12, Suppl. 1, S17-S-23

Moharami et al. 2014. Effect of TiO2, Al2O3, and Fe3O4 Nanoparticles on Phosphorus Removal from Aqueous Solution. Environ. Prog. Sustain. Energy 33, 1209-1219