Titanium nitride

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 17, 2015 to November 19, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

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 (48 ± 1 hour), final water quality and D. magna immobility assessments were 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 48-hour-old test solutions were conducted in order to evaluate the range of exposures of soluble test materials as represented by dissolved titanium concentrations.
Control and dilution water:
Reconstituted moderately hard laboratory water (USEPA, 2002), adjusted to pH 6.0, 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 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 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, which also used a synthetic fiber filter as used in this study. 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), and the base water was de-ionized water to which reagent-grade salts were added per USEPA (2002). 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.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: N.a.
- Source: In-house cultures maintained at Ramboll Environ’s test facilities in Brentwood, Tennessee.
- Age at study initiation (mean and range, SD): Neonates, less than 24h
- Feeding during test: No

ACCLIMATION
- Acclimation period: Not applicable; culture conditions same as test conditions
- Acclimation conditions (same as test or not): Yes (temperature, culture water, and photoperiod and light intensity)
- Type and amount of food/feeding frequency: Feeding of D. magna while in culture and feeding of neonates immediately prior to the test (minimum of two hours prior to test initiation) consisted of feeding a green algae (Pseudokirchneriella subcapitata) and Cerophyll suspension (combined total solids concentration of 1,700 to 1,900 mg/L) at a rate of 60 mL/L in culture and 17 mL/L immediately prior to neonate testing.
- Health during acclimation (any mortality observed): Information related to test organism health while in culture was documented and included notations regarding parent-organism mortality, absence of ephippia, and numbers of neonates produced.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

81.6 mg CaCO3/L

Test temperature:

21.7-22.0 °C

pH:

5.93-7.89

Dissolved oxygen:

7.9-8.6 mg/L

Salinity:

<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: borosilicate glass beakers (60 mL)
- Type (delete if not applicable): loosely covered with clear plastic sheets
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM/WATER PARAMETERS
- Source/preparation of dilution 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 organisms (including D. magna), and is a suitable water for OECD guideline 202.
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.
- Alkalinity: 45.0 mg/L
- Conductivity: 195-295 µS/cm
- Culture medium different from test medium: no
- 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: Yes, to pH 6
- Photoperiod: 16 hr light, 8 hr dark
- Light intensity: 997 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mobility, at 24-hr intervals

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

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

See section "any other information on 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 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 was 6.98). 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 Schafers (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.

 

Tab. 1: Experimental Design and Measured Titanium Concentrations

Titanium Nitride Loading  Concentration (mg/L)	Initial Dissolved Titanium Concentration (µg/L)	Final (48 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 in the presence of 100 mg/L titanium nitride by repeated acid additions and pH readjustment of the test water to pH 6 followed by an extended gas equilibration period and further 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 was 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. The extended acid readjustment resulted in an approximately four-fold increase in toxicity test water specific conductance values. Thus, this follow up testing confirmed that extended acid readjustment of toxicity test waters would have resulted in a marked shift in the ionic composition of the standard test water specified in the test protocol. The extended pH adjustment would also have resulted in a change in dissolved solids concentration in the highest titanium nitride exposure that would not be present in other exposures, resulting in an additional test variable within the toxicant exposure series. 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.

 

Water quality

All physical and chemical parameters for the study were within targeted ranges and therefore suitable for toxicity testing of D. magna (Table 2). The measured test and control water temperatures ranged from 21. 7 to 22.0 °c, which is within the OECD-specified test range of 18 to 22 °c and ± 1 °C for a given test. Dissolved oxygen measurements ranged from 7.9 to 8.6 mg/L, and pH ranged from 5.93 s.u. to 7.89. 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 control solutions in which test organism immobility was minimal and therefore did not impact toxicity test results. The lower pH conditions than preferred in the OECD protocol (pH 6 to 9) was necessitated by the need for acidic conditions in the dissolution test and an appropriate control in the low-pH toxicity test that was also intended to maximize product toxicity. 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 total hardness value for the test water was 81.6 mg CaC03/L, and total alkalinity was 45 mg/l (Table 3) upon test water preparation. The lower hardness than conventionally used in D. magna toxicity testing (although well within their range of tolerance) was due to the same conditions being present in the D. magna cultures from which the toxicity test organisms were obtained. This lower 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 997 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 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 – parameters measured daily in titanium nitride test

Nominal Titanium Nitride Concentration (mg/L)	pH (s.u.)	Dissolved Oxygen (mg/L)	Specific Conductance (μS/cm)	Temperature (°C)
0 (unadj. pH)	7.75 to 8.40	8.2 to 8.6	195 to 287	22.0 to 22.0
0 (pH 6)	5.93 to 7.90	7.9 to 8.5	200 to 260	21.7 to 22.0
6.25	6.01 to 7.89	8.1 to 8.4	207 to 291	22.0 to 22.0
12.5	6.40 to 7.82	8.1 to 8.6	214 to 295	21.7 to 21.9
25	6.47 to 7.74	8.0 to 8.6	218 to 287	21.9 to 22.0
50	6.51 to 7.70	8.1 to 8.4	211 to 292	21.8 to 22.0
100	6.79 to 7.79	8.2 to 8.5	215 to 286	21.8 to 22.0

 

Table 3. 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	81.6	45.0	< 0.02

(a) Parameters were measured in all batches of test water used in this study.

Biological results

A maximum combined-replicate average of 10 percent Daphnia magna immobility was observed in the 100 mg/L loading rate titanium nitride exposure. This was identical to that observed in the pH 6.0 control exposure that lacked previous titanium nitride addition. Product addition therefore resulted in no demonstrable toxicity to D. magna. An EC₅₀ 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 4. Daphnia magna immobilisation data

Treatment Group Titanium Nitride	Rep ID	Initial Number Exposed	Number Immobilized (24 hr. /48 hr.)	Mean Number Immobilized (24 hr. /48 hr.)*	Percent Immobilized (24 hr. /48 hr.)	Mean Percent Immobilized (24 hr. /48 hr.)*
pH 6 Control	A B C D	5 5 5 5	0/1 0/1 0/0 0/0	0.1	5 5 0 0	0/10
6.25 mg/L	A B C D	5 5 5 5	0/0 0/0 0/0 0/0	0	0 0 0 0	0/0
12.5 mg/L	A B C D	5 5 5 5	0/0 0/0 0/0 0/0	0	0 0 0 0	0/0
25 mg/L	A B C D	5 5 5 5	0/0 0/0 0/0 0/0	0	0 0 0 0	0/0
50 mg/L	A B C D	5 5 5 5	0/1 0/0 0/0 0/0	0.05	5 0 0 0	0/5
100 mg/L	A B C D	5 5 5 5	0/1 0/1 0/0 0/0	0.1	5 5 0 0	0/10

Validity criteria fulfilled:

yes

Conclusions:

Titanium nitride is not toxic to D. magna after 48 h exposure at a nominal concentration of 100 mg/L.

Executive summary:

In a 48-h acute toxicity study according to OECD guideline 202 (GLP), Daphnia magna 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 demonstrated a lack of acute toxicity to D. magna after 48-hour exposure.

No mortality was observed in 100 mg/L loading rate exposures, identical to that observed in the control exposures. An acute EC₅₀ 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 D. magna in accordance with the classification system of CLP.

Description of key information

Titanium nitride is not toxic to Daphnia magna under the conditions of OECD guideline 202.

Additional information

In a 48-h acute toxicity study according to OECD guideline 202 (GLP), Daphnia magna 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. 

As 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, effect values were based on nominal loading rates.

Titanium nitride did not show acute toxicity in D. magna after 48-hour exposure at concentrations up to 100 mg/L. Thus, an acute EC₅₀ value of greater than 100 mg/L titanium nitride (nominal) was determined in this study.

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