Titanium nitride

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 28, 2015 to December 9, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

The measured test and control water temperatur range was lower (20.3-21.7 ºC) than specified (21-25 ºC). pH was lower than in the OECD guideline (pH 6 to 9 s.u.). This did not influence the overall outcome of this test. For details see below.

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

Details on sampling:

At test termination (96 hours), final water quality assessment was conducted and test solutions preserved for titanium analyses.
At test termination, approximately 50 mL of test solution was removed from each of the four control vessels and four exposure concentrations for a combined total of 200 mL used for titanium analyses.

Vehicle:

no

Details on test solutions:

Following the seven-day dissolution exposure, the test solutions were prepared by diluting the maximum dissolution exposure with the appropriate pH 6.0 test water to achieve the suitable range of product exposure concentrations identified in the range-finding toxicity test. Titanium analyses of the initially-prepared test solutions and 96-hour-old test solutions were conducted in order to evaluate the range of exposures of soluble test material as represented by dissolved titanium concentrations.

Control and dilution water:
Reconstituted moderately hard laboratory water (USEPA, 2002), adjusted to pH 6.0 s.u., served as the diluent and primary control water. A secondary moderately hard water control (unadjusted pH) was evaluated to assess test organism mortality in the event effects were observed in the primary control water. The test water was prepared from de-ionized water to which reagent-grade salts were added per USEPA (2002). This water is commonly used in the culture and testing of a variety of freshwater organisms (including the fathead minnow), and is a suitable water for OECD guideline 203. The test water is also routinely confirmed to be free of contaminants such as pesticides and heavy metals, and confirmed to contain total organic carbon (TOC) concentrations of less than 1 mg/L.

Test substance preparation and dosing:
The dissolution procedure followed guidance provided in the OECD Series on Testing and Assessment Publication Number 29 for transformation/dissolution testing (OECD, 2001). The dissolution test medium was moderately hard reconstituted water prepared according to USEPA (2002) with the pH adjusted to 6.0 s.u. with reagent grade 1 N hydrochloric acid prior to test material addition. The test solution was then prepared by establishing a 100 mg/L (nominal) 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) to remove undissolved particles. This filter pore size is the same as that used in the Klawonn (2015) Titanium Nitride transformation/dissolution study (see IUCLID section 4.8). By filtering the sample, only dissolved product materials’ toxicity was assessed in the solutions evaluated.
Toxicity testing of solutions was conducted by dilution of the 100 mg/L dissolution preparation to establish exposures of the nominal test material loading rates of 6.25, 12.5, 25, 50, and 100 mg/L whole product.

Test solution preparation was conducted at temperatures of 24 ± 1 °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 toxicity tests (adjusted to pH 6.0 s.u.), and the base water was de-ionized water to which reagent-grade salts were added per USEPA (2002). “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.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Aquatic BioSystems (ABS), Inc. (Fort Collins, Colorado, USA)
- Length at study initiation: Average length 1.9 cm (range 1.6-2.0 cm)
- Weight at study initiation: Average wet weight 0.100 g (range 0.0614-0.1653 g)
- Method of breeding: Not applicable
- Feeding during test: No

ACCLIMATION
- Acclimation period and conditions: De-chlorinated tap water with pH, hardness, and alkalinity very similar to that of the test water (i.e., Environmental Protection Agency (USEPA) reconstituted moderately hard water) for 13 days prior to testing; organisms were acclimated to the test dilution water (i.e., USEPA moderately hard water) at 21 °C for 48 hours prior to toxicity test initiation.
- Type and amount of food:
- Feeding frequency: No data; fish were not fed for 24 hours prior to test initiation
- Health during acclimation: Good health (i.e., zero mortality, and no unusual behavioural conditions or anomalies were observed)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

85.6 mg CaCO3/L

Test temperature:

20.3-21.7 °C

pH:

6.06-7.67 s.u.

Dissolved oxygen:

7.9-8.6 mg/L

Salinity:

Total residual chlorine: < 0.02 mg/L

Nominal and measured concentrations:

Nominal: 0 (control), 6.25, 12.5, 25, 50, and 100 mg/L TiN
Test endpoints based on nominal initial loading rates.

Details on test conditions:

TEST SYSTEM
- Test vessel: 4 L inert plastic vessels
- Type: Open; test chambers were loosely covered with clear plastic sheets that allowed proper light penetration and gas exchange while minimizing test solution evaporation.
- Fill volume: 3 L
- Aeration: Yes, continously (dissolved oxygen concentrations were maintained at > 8 mg/L)
- Renewal rate of test solution (frequency/flow rate): n.a., static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): n.a.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: standard reconstituted freshwater
- Total organic carbon: < 1 mg/L
- Metals: Free of heavy metals
- Pesticides: Free of pesticides
- Chlorine: < 0.02 mg/L
- Alkalinity: 49 mg/L CaCO3
- Conductivity: 205-257 µS/cm
- Intervals of water quality measurement: Test solution pH, conductivity, temperature, and dissolved oxygen were monitored at 24-hour intervals throughout the study. Test water residual chlorine, total hardness, and total alkalinity were documented at test initiation.

OTHER TEST CONDITIONS
- Adjustment of pH: Reconstituted moderately hard laboratory water (USEPA, 2002), adjusted to pH 6.0, served as the diluent and primary control water
- Photoperiod: 16 hr light 8 hr dark
- Light intensity: 564 lux

EFFECT PARAMETERS MEASURED:
Observations of fish mortality, loss of equilibrium, immobility, changes in pigmentation, etc. in each test chamber were made at 24, 48, 72, and 96 hours.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: See section "Nominal and measured concentrations" above
- A pH-adjusted (pH 6.0 s.u.) and pH unadjusted moderately hard water control was evaluated in this testing.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

See section "Any other information on results incl. tables".

Sublethal observations / clinical signs:

Analytical results

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 toxicity 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 despite the seven-day exposure period to the toxicity test water. This differs somewhat from a previous Titanium Nitride dissolution study (Klawonn, 2015; see IUCLID section 4.8) in much softer, lower ionic strength waters than used in this study. The Klawonn (2015) study was conducted in a much lower hardness and lower alkalinity dissolution medium than used in this study because such a medium as used by Klawonn (2015) would be unsuitable as a toxicity test medium. This was further evaluated as discussed below.

Table 1. Experimental Design and Measured Titanium Concentrations
Titanium Nitride Loading   Concentration (mg/L)	Initial Dissolved Titanium Concentration (µg/L)	Final (96 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

 

Additional dissolution testing was conducted in a follow-up study in which moderately hard toxicity test water was maintained at pH 6.0 s.u. in the presence of 100 mg/L titanium nitride by repeated acid addition and pH readjustment of the test water to pH 6 s.u. 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 s.u., 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 D. magna 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. 

 

Water Quality

All physical and chemical parameters for the study were within targeted ranges and therefore suitable for fathead minnow toxicity testing (Tables 2 through 5). The measured test and control water temperatures ranged from 20.3 to 21.7 ºC, which is at the lower end of the 21 to 25 ºC temperature range specified by the protocol, but is well within the range of test conditions known to be acceptable for toxicity testing of P. promelas. Therefore, this deviation is considered to be of minor importance and has no impact on the validity of the results. Dissolved oxygen concentrations ranged from 7.9 to 8.6 mg/L, and pH ranged from 6.06 s.u. to 7.67 s.u. 

 

Some drift in test solution pH was observed as a result of gas equilibration of test solutions following removal from the dissolution test apparatus and dilution with the pH 6.0 dilution water. 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. The drift in pH is not unusual in such tests, and some of the highest drift was observed in the nominal 100 mg/L product loading rate exposure (pH 6.06 to 6.80 s.u.). Given the absence of toxicity in all test and control exposures, this pH drift was not a significant source of test organism stress and therefore did not impact toxicity test results. The lower pH conditions than preferred in the OECD protocol (pH 6 to 9 s.u.) were necessitated by the need for acidic conditions in the dissolution procedure. The total hardness value for the test water was 85.6 mg CaCO₃/L, and total alkalinity was 49 mg/L (Table 6) upon test water preparation. Light intensity was 564 lux, and 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 Method 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.    

Table 2. Range of Water Quality Conditions – Parameters MeasuredDaily in Titanium Nitride Test
Nominal Titanium Nitride Concentration (mg/L)	pH (s.u.)	Dissolved Oxygen (mg/L)	Specific Conductance (µS/cm)	Temperature (^oC)
0 (unadj. pH)	7.41 to 7.67	8.0 to 8.4	206 to 220	21.0 to 21.7
0 (pH 6)	7.36 to 7.76	7.9 to 8.5	205 to 227	21.0 to 21.3
6.25	7.32 to 7.58	8.2 to 8.4	213 to 224	20.8 to 21.2
12.5	7.29 to 7.54	8.2 to 8.6	214 to 226	20.7 to 21.4
25	7.26 to 7.50	8.1 to 8.5	217 to 229	21.0 to 21.3
50	7.24 to 7.47	8.0 to 8.5	215 to 244	20.3 to 21.2
100	6.06 to 6.87	8.1 to 8.5	239 to 257	20.4 to 21.3

 

Table 3.Dissolved Oxygen Concentrations During Titanium     Nitride Fathead Minnow Acute Mortality Test at 24-hour Observation Periods (mg/L)
Nominal Titanium Nitride C (mg/L)	Test Initiation	24 Hours	48 Hours	72 Hours	96 Hours^(a)
0 (unadj. pH)	8.3	8.5	8.4	7.9	8.4
0 (pH 6)	8.3	8.2	8.0	8.4	8.2
6.25	8.4	8.4	8.3	8.2	8.2
12.5	8.4	8.4	8.3	8.2	8.6
25	8.5	8.3	8.5	8.1	8.5
50	8.5	8.0	8.3	8.1	8.3
100	8.5	8.1	8.1	8.2	8.1
(a) Test termination.

 

Table 4. pH During Titanium Nitride Fathead Minnow Acute               Mortality Test at 24-hour Observation Periods (s.u.)
Nominal Titanium Nitride C (mg/L)	Test Initiation	24 Hours	48 Hours	72 Hours	96 Hours^(a)
0 (unadj. pH)	7.67	7.58	7.47	7.41	7.56
0 (pH 6)	7.39	7.76	7.36	7.42	7.59
6.25	7.58	7.45	7.32	7.35	7.55
12.5	7.54	7.38	7.29	7.31	7.50
25	7.50	7.32	7.26	7.28	7.44
50	7.47	7.31	7.24	7.25	7.40
100	6.80	6.87	6.85	6.38	6.06
(a) Test termination.

 

 

Table 5. Temperature During Titanium Nitride Fathead Minnow Acute Mortality Test at 24-hour Observation Periods (°C)
Nominal Titanium Nitride C (mg/L)	Test Initiation	24 Hours	48 Hours	72 Hours	96 Hours^(a)
0 (unadj. pH)	21.7	21.0	21.0	20.3	21.3
0 (pH 6)	21.0	21.0	21.0	21.0	21.3
6.25	21.2	21.1	21.0	20.8	20.9
12.5	21.4	21.0	21.2	20.7	20.8
25	21.3	21.0	21.1	21.0	21.0
50	21.2	21.1	21.0	20.3	20.3
100	21.3	21.0	21.0	20.4	20.3
(a) Test termination.

                                  

 

Table 6. Range of Water Quality Conditions – Parameters Measured Less Than Daily in Titanium Nitride Test
Treatment Group	Total Hardness^(a) (mg/L CaCO3)	Total Alkalinity^(a) (mg/L CaCO3)	Total Residual Chlorine^(a) (mg/L)
T0 - Control	85.6	49.0	< 0.02
(a)  Parameters were measured in the test water used in this study.

 

Biological Results

No fathead minnow mortality was observed in the 100 mg/L loading rate titanium nitride exposure (Table 7). This was identical to that observed in the control exposures that lacked titanium nitride addition. Product addition therefore resulted in no demonstrable toxicity to the fathead minnow. An LC₅₀ value of greater than 100 mg/L titanium nitride was determined. These results are not unexpected given the lack of toxicity observed in the range-finding test at 1,000 mg/L titanium nitride.

Table 7.Pimephales promelasMortality Data
Treatment Group Titanium Nitride	Rep ID	Initial Number Exposed	Number Dead (48/96 hour)	Mean Number Dead (48/96 hour)*	Percent Mortality (48/96 hour)*	Mean Percent Mortality (48/96 hour)*
pH 6 Control	A B	5 5	0/0 0/0	0/0 0/0	0/0 0/0	0/0 0/0
6.25 mg/L	A B	5 5	0/0 0/0	0/0 0/0	0/0 0/0	0/0 0/0
12.5 mg/L	A B	5 5	0/0 0/0	0/0 0/0	0/0 0/0	0/0 0/0
25 mg/L	A B	5 5	0/0 0/0	0/0 0/0	0/0 0/0	0/0 0/0
50 mg/L	A B	5 5	0/0 0/0	0/0 0/0	0/0 0/0	0/0 0/0
100 mg/L	A B	5 5	0/0 0/0	0/0 0/0	0/0 0/0	0/0 0/0
*Combined replicates.

Validity criteria fulfilled:

yes

Remarks:

dissolved oxygen concentrations in the control exposures above 60 percent saturation (5.2 mg/L at 22 °C), no control organism mortality or signs of stress, constant test conditions

Conclusions:

TItanium nitride is not toxic to fathead minnow after 96h exposure at a nominal concentration of 100 mg/L.

Executive summary:

In a 96-h acute toxicity study according to OECD guideline 203 (GLP), fathead minnow (Pimephales promelas) 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 limits of quantification (2 µg/L) at toxicity test initiation and at test termination in all control and test exposures.

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

Titanium nitride did not exert acute toxicity on fathead minnow during 96-hour exposure. No mortality was observed in 100 mg/L loading rate exposures, identical to that observed in the control exposures. An acute LC₅₀ value of greater than 100 mg/L titanium nitride (nominal) was determined from this study.

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

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

Results synopsis

Test organism size/age: Average length 1.9 cm, average wet weight 0.100 g

Test type: Static

LC₅₀: > 100 mg/L TiN (nominal)

Endpoint(s) effected: Mortality

Description of key information

Titanium nitride is not toxic to fathead minnow after 96 h exposure at a nominal concentration of 100 mg/L.

Additional information

In a 96-h acute toxicity study according to OECD guideline 203 (GLP), fathead minnow (Pimephales promelas) 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. 

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 (mortality) was determined using the nominal loading rate of the test material.

No mortality was observed in 100 mg/L loading rate exposures, identical to that observed in the control exposures. An acute LC₅₀ value of greater than 100 mg/L titanium nitride (nominal) was determined in this study.