Titanium nitride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Product dissolution initiated November 28, 2015. Toxicity test initiated December 5, 2015 and terminated December 8, 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment Publication Number 29 for transformation/dissolution testing (OECD, 2001)

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment Number 23 for Testing of difficult substances and mixtures (OECD, 2000)

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The dissolution procedure followed guidance provided in the OECD Series on Testing and Assessment Publication Number 29 for dissolution testing (OECD, 2001). The dissolution test medium was USEPA algae culture and test medium prepared according to USEPA (2002) with the pH adjusted to 6.0 with reagent-grade 1 N hydrochloric acid prior to test material addition. The test solution was then prepared by establishing a 100 mg/L preparation of TiN in the test medium. Following a seven-day dissolution (elution) exposure at 100 revolutions per minute on a laboratory shaker table, solutions were filtered (0.2 micron nylon filter) to remove undissolved particles. This filter pore size is the same as that used in the Klawonn (2015) titanium nitride dissolution study. By filtering the sample, only dissolved product materials’ toxicity was assessed in the solutions evaluated in P. subcapitata toxicity tests.
Test solution preparation was conducted at temperatures of 23.7 to 24.3 °C. Test solution preparation vessels were pre-cleaned 1 L borosilicate amber glass bottles sealed with Teflon-lined plastic lids. The dissolution water was the same as that used in the P. subcapitata toxicity test (adjusted to pH 6.0), and the base water was de-ionized water to which reagent-grade salts and nutrients sufficient for growth of P. subcapitata were added per USEPA (2002). Similarly prepared dissolution test water TOC concentrations have been confirmed in previous testing to be below levels of detection (less than 1 mg/L). “Surrogate” test vessels, identical to those used for test material exposure, were established exclusively to monitor test solution pH, temperature, and dissolved oxygen during the dissolution exposure.
Titanium analyses of the initially prepared test solutions and 72-hour-old test solutions were conducted in order to evaluate the range of exposures of soluble test materials as represented by dissolved titanium concentrations.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): In-house cultures maintained at Ramboll Environ’s test facilities in Brentwood, Tennessee; original cultures were obtained from a commercial supplier with appropriate taxonomic documentation.
- Age of inoculum (at test initiation): log-phase P. subcapitata from a culture of known cell density were added to the control and test exposures at test initiation
- Method of cultivation: P. subcapitata are continuously cultured at Ramboll Environ in the USEPA algae growth medium recommended for culture and testing of algae (USEPA, 2002). Algae culture conditions were identical to the toxicity test conditions in terms of temperature, culture water, photoperiod, and light intensity.

ACCLIMATION
- Acclimation period: N.a.
- Culturing media and conditions (same as test or not): Same
- Any deformed or abnormal cells observed: None reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

16.8 mg/L CaCO3

Test temperature:

22.7-24.2 °C

pH:

6.92-8.54

Dissolved oxygen:

7.4-8.4 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 6.25, 12.5, 25, 50, and 100 mg/L titanium nitride C

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL clear glass Erlenmeyer flasks
- Type: Flasks were sealed with cotton balls to allow gas exchange but minimize test solution evaporation and entry of dust or particles into test solutions
- Fill volume: 100 ml
- Aeration: No
- Initial cells density: 1.00E+04 cells/ml
- Control end cells density: 6.44E+05 cells/ml (mean value)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (pH-adjusted and unadjusted)

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM/WATER PARAMETERS
- Preparation of dilution water: From de-ionized water to which reagent-grade salts and nutrients were added per USEPA (2002)
- Total organic carbon: < 1 mg/L
- The de-ionized water used to prepare the test media is routinely confirmed to be free of contaminants such as pesticides and heavy metals.
- Alkalinity: 16.0 mg/L CaCO3
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: Yes (pH 6.0)
- Photoperiod: 24:0 h light:dark
- Light intensity and quality: Targeted light intensity of approximately 4,300 to 8,600 lux; light intensity at the water surface was documented at test initiation and test termination

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Direct cell density counts were conducted with a hemocytometer at test termination in all control and test (titanium nitride) replicates

TEST CONCENTRATIONS
- Range finding study: Yes (100 and 1000 mg test substance/L)
- Test concentration, definitive study: 6.25, 12.5, 25, 50, and 100 mg/L were obtained by dilution of the 100 mg/L dissolution preparation; dilution was conducted with pH 6-adjusted algae growth medium

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The experimental conditions utilized in this study maintained dissolved oxygen concentrations in the control exposures above 60 percent saturation (5.2 mg/L at 22 °C), and consistently maintained the pH of the test waters between 6.9 and 8.5. Average cell density in the pH-adjusted control exposures was 5.15E+05 cells/mL following the 72-hour exposure period, meeting the minimum cell growth requirement of a 16-fold increase relative to the test inoculation density of 1.0E+04 cells/mL. No signs of stress, cell agglomeration, or cell discoloration were observed in any test or control exposure. The minimum test precision requirement of a coefficient of variation of less than 35 percent for section-by-section growth rates was also met (maximum coefficient of variation for any 24-hour growth period was 21.3 percent in the non-pH adjusted control exposures on which this criterion is based). The average 72-hour coefficient of variation for growth rate in the pH-adjusted and non-adjusted control exposures was less than three percent compared to a maximum allowable coefficient of variation of seven percent. Thus, adequate test conditions were maintained, and all test acceptability criteria and other methodologies specified in the study protocol were met.

Reported statistics and error estimates:

P. subcapitata yield, expressed as cell density (cells/mL as determined by direct count), was assessed for the 72 hour exposure period with the ToxCalc™ version 5.0.23A statistical package (TIDEPOOL Scientific Software). P. subcapitata specific growth rate (SGR), determined by log-normal transformation of cell density for the control and titanium nitride exposures for the 72-hour test period, was also evaluated. In both cases (cell yield and SGR) test endpoints were evaluated by hypothesis testing to determine the NOEC value, and point estimate techniques were used to determine the EC50 value. All endpoints were determined relative to the pH-adjusted control exposures.

1.     Analytical Results

1.1     Loading Rates

Table 1 summarizes the results of the dissolved titanium analyses on the control and titanium nitride toxicity test exposures. Measured titanium concentrations were below the limits of quantification (2 µg/L) at test initiation and at test termination in all control and test exposures. The absence of dissolved titanium in test solutions at test initiation indicates that titanium was not readily leached from Titanium Nitride C during the seven-day exposure period to the toxicity test water. It should be noted, however, that although acidic conditions were maintained in the dissolution test medium, dissolution pH drifted above the initial pH 6.0 condition following product addition (maximum dissolution pH during the seven day exposure period was 6.92). As compared to the results of other dissolution tests conducted with titanium nitride (Klawonn, 2015), the pH condition in this study may have contributed to the absence of detectable titanium concentrations in the dissolution test medium subsequently used in toxicity testing. In the Klawonn (2015) study, pH 6 conditions were better maintained in a much lower hardness and lower alkalinity dissolution medium which would be unsuitable as a toxicity test medium.

Additional dissolution testing was conducted in a follow-up study in which moderately hard toxicity test water was maintained at pH 6.0 in the presence of 100 mg/L titanium nitride by repeated acid addition and pH readjustment of the test water to pH 6 followed by an extended equilibration period and pH readjustment. Following a seven-day dissolution test in which test solution pH was maintained between 5.82 and 6.27, measured dissolved titanium concentrations were less than 2 µg/L. This confirmed that the minimal dissolution of titanium in the dissolution exposures used in toxicity testing were not an artifact of the higher pH conditions in the toxicity test waters used as the dissolution test medium, and demonstrated that titanium nitride is not soluble in higher ionic strength, higher hardness test waters such as those needed for aquatic toxicity testing. This follow up testing also confirmed that extended acid readjustment of toxicity test waters could result in ion imbalance in the test solution that could have resulted in additional stress to test organisms. The extended acid readjustment resulted in an approximately four-fold increase in toxicity test water specific conductance values. Additionally, the procedures used in this study provide a higher degree of environmental and ecological relevance with respect to conditions under which P. subcapitata would be present and environmental exposures could occur. Higher hardness and alkalinity in test waters would influence titanium solubility in addition to pH and other factors such as temperature. Further evaluations of the role of water quality conditions in altering titanium solubility when associated with titanium nitride could be conducted to evaluate the relative influence of various factors, but the titanium associated with titanium nitride was of very limited solubility when assessed under these ecologically realistic conditions, and toxicity test conditions that must be maintained in order to ensure test organism health. 

Table 1. Experimental Design and Measured Titanium Concentrations

Titanium Nitride Loading Concentration (mg/L)	Initial Dissolved Titanium Concentration (µg/L)	Final (72 hr.) Dissolved Titanium Concentration (µg/L)
0 (unadj. pH)	< 2.0	< 2.0
0 (pH 6)	< 2.0	< 2.0
6.25	< 2.0	< 2.0
12.5	< 2.0	< 2.0
25	< 2.0	< 2.0
50	< 2.0	< 2.0
100	< 2.0	< 2.0

 

1.2     Water quality

All physical and chemical parameters for the study were within targeted ranges and therefore suitable for toxicity testing of P. subcapitata (Table 2). The measured test and control water temperatures ranged from 22.7 to 24.2 ºC, which is within the protocol-specified test range of 21 to 24 ºC and ± 2 °C. Dissolved oxygen measurements ranged from 7.4 to 8.4 mg/L, and pH ranged from 6.92 to 8.54. The variation in pH was likely due to gas equilibration with the test medium following removal from the sealed dissolution test containers and placement in the covered toxicity test vessels that are designed to allow natural gas exchange. Additionally, as P. subcapitata cells grew and cell density increased, test solution pH would be increased as a result of photosynthetic activity (i.e., removal of CO₂ from test solutions, decreasing carbonic acid concentrations). The drift in pH is not unusual in such tests, and slightly higher pH drift was observed in control solutions in which test organisms growth was not impeded. The pH drift therefore did not impact toxicity test results. The total hardness value for the test water was 16.8 mg CaCO₃/L, and total alkalinity was 16.0 mg/L (Table 3) upon test water preparation. This is consistent with the soft-water test conditions necessitated by the use of the standard algae growth and test medium. This low water hardness would also have enhanced product solubility in the dissolution tests. The lower pH and somewhat softer water conditions were also maintained in control exposures, and as demonstrated by acceptable control organism response, did not impact test quality. Light intensity was 5,479 lux at test initiation and 5,511 lux at test termination. Total residual chlorine (TRC) was below levels of detection (Table 3). Although the TRC detection limit of 0.02 mg/L is somewhat higher than the desired limit per OECD guideline 202, the test water was prepared from distilled water that is known to be free of contaminants, and was of acceptable quality for toxicity testing based on acceptable control organism response.    

Table 2. Range of water quality conditions in titanium nitride test (a)

Nominal Titanium Nitride Concentration (mg/L)	pH	Dissolved Oxygen (mg/L)	Specific Conductance (μS/cm)	Temperature (°C)
0 (unadj. pH)	7.67 to 8.54	7.4 to 8.3	102 to 120	23.1 to 24.0
0 (pH 6)	6.93 to 7.95	7.9 to 8.4	103 to 118	22.8 to 23.3
6.25	7.30 to 8.01	7.5 to 8.0	101 to 118	23.1 to 23.6
12.5	7.14 to 7.90	7.7 to 8.3	102 to 117	23.2 to 24.1
25	7.06 to 7.72	7.6 to 8.3	101 to 117	22.7 to 23.2
50	7.02 to 8.31	7.8 to 8.4	102 to 121	23.0 to 23.4
100	6.92 to 8.33	7.9 to 8.3	102 to 119	23.0 to 24.2

(a)Dissolved oxygen and conductivity measured at test initiation and test termination, pH and temperature measured at test initiation and at 24-hour intervals until test termination.

Table 3. Water quality conditions – parameters measured at test initiation

Treatment Group	Total Hardness^(a) (mg/L CaCO3)	Total Alkalinity^(a) (mg/L CaCO3)	Total Residual Chlorine^(a) (mg/L)
T0 - Control	16.8	16.0	< 0.02

1.3     Biological Results

An average cell density (yield) of 5.15 × 10⁵cells/mL was observed in the pH-adjusted control exposures for the 72-hour exposure period. The average cell density in the titanium nitride exposures ranged from 4.45 × 10⁵ to 6.00 × 10⁵ cells/mL for the same exposure period, with the highest average cell density observed in the highest nominal (100 mg/L) titanium nitride loading rate. The resulting yield-based EC₅₀ and NOEC values were therefore greater than 100 mg/L and 100 mg/L, respectively. Similar results were obtained on the SGR basis assessment. The log-normal transformed average SGR for the pH 6 control exposure was 1.31 cells/day compared to a range of 1.26 to 1.36 cells/day in the titanium nitride exposures, with the highest SGR observed in the 100 mg/L titanium nitride loading rate exposure. Regardless of the basis of toxicity determination (yield or SGR), product addition resulted in no demonstrable toxicity to P. subcapitata. These results are not unexpected given the lack of toxicity observed in the range-finding test at a nominal 1,000 mg/L titanium nitride concentration. Table 4. P. subcapitata cell density and SGR (72 hour) 

Treatment Group Titanium Nitride	Rep ID	Initial Number Exposed (cells/mL)	Number of Cells/Replicate	Mean Number of Cells (cells/mL) / SGR (cells/d)	Mean Change vs. Control (cells/mL)	Mean Percent Change vs. Control (yield/SGR)
pH 6 Control	A B C D E F	1 x 10^4 1 x 10^4 1 x 10^4 1 x 10^4 1 x 10^4 1 x 10^4	4.95 x 10^5 5.20 x 10^5 4.75 x 10^5 4.60 x 10^5 5.05 x 10^5 6.35 x 10^5	5.15 x 10^5/1.31	NA	NA
6.25 mg/L	A B C	1 x 10^4 1 x 10^4 1 x 10^4	4.35 x 10^5 6.00 x 10^5 5.30 x 10^5	5.22 x 10^5/1.32	0.067 x 10^5	1.3/0.7
12.5 mg/L	A B C	1 x 10^4 1 x 10^4 1 x 10^4	4.30 x 10^5 5.20 x 10^5 5.00 x 10^5	4.83 x 10^5/1.29	-0.317 x 10^5	-6.1/-0.3
25 mg/L	A B C	1 x 10^4 1 x 10^4 1 x 10^4	4.15 x 10^5 4.35 x 10^5 4.85 x 10^5	4.45 x 10^5/1.26	-0.700 x 10^5	-13.6/-0.7
50 mg/L	A B C	1 x 10^4 1 x 10^4 1 x 10^4	4.55 x 10^5 4.90 x 10^5 4.70 x 10^5	4.72 x 10^5/1.28	-0.433 x 10^5	-8.4/-0.4
100 mg/L	A B C	1 x 10^4 1 x 10^4 1 x 10^4	6.00 x 10^5 5.90 x 10^5 6.10 x 10^5	6.00 x 10^5/1.36	0.850 x 10^5	16.5/0.9

NA = Not applicable

Validity criteria fulfilled:

yes

Conclusions:

TiN is not toxic to P. subcapitata. Based on the nominal concentration, the 72-hour ErC50 is > 100 mg/L, the NOEC based on the grwoth rate is 100 mg/L.

Executive summary:

In a 72-h toxicity study according to OECD guideline 201 (GLP), algae (Pseudokirchneriella subcapitata) were exposed to titanium nitride at nominal concentrations of 0 (control), 6.25, 12.5, 25, 50 and 100 mg TiN/L under static conditions. 

The dissolution test system for preparing test solutions adhered to guidelines outlined in the OECD Series on Testing and Assessment Number 29 (OECD, 2001). General practices for testing of difficult to test substances as outlined in OECD Series of Testing and Assessment Number 23 (OECD, 2000) were also incorporated as applicable.

Measured titanium concentrations were below the limit of quantification (2 µg/L) at toxicity test initiation and at test termination in all control and test exposures.

Thus, the test endpoint (growth rate) was determined using the nominal loading rate of the test material.

Titanium nitride did not exert any toxicity on P. subcapitata during 72-hour exposure.

No inhibition of algal growth was observed in 100 mg/L loading rate exposures, identical to that observed in the control exposures. An acute E_rC₅₀ value of greater than 100 mg/L titanium nitride (nominal) was determined from this study. The NOEC based on the growth rate is 100 mg/L.

Based on the results of this study, titanium nitride would not be classified as toxic to P. subcapitata in accordance with the classification system of CLP.

This toxicity study is classified as acceptable and satisfies the guideline requirement for an aquatic toxicity study to algae.

Results synopsis:

Test organism: Pseudokirchneriella subcapitata

Test type: static

72 hr E_rC₅₀:  > 100 mg/L

72 hr NOEC:  100 mg/L

Endpoint(s) effected:  Growth rate

Description of key information

Under the conditions of OECD guideline 201 the test substance titanium nitride is not toxic to algae (P. subcapitata).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to titanium nitride at nominal concentrations of 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L under static conditions in accordance with OECD guideline 201.  The NOEC and EC50 values based on growth rate were 100 mg/L and > 100 mg/L, respectively.

This toxicity study is classified as acceptable and satisfies the guideline requirements for a study investigating the aquatic acute toxicity to algae.