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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27 Nov 2012 - 21 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Pre-study solubility work showed that the test item was insoluble in water using the traditional methods of preparation, e.g. ultrasonication. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

- Method of the media preparation trial: An amount of test item (250 mg) was added to the surface of 5 liters of reconstituted water and stirred vigorously using a magnetic stirrer for 10 minutes. The pH of the media was adjusted to 7.8 and the stirring restarted for a further 24 hours. After stirring, any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (initial 500 mL discarded) to give the 100% v/v saturated test solution from which serial dilutions were made to obtain the remaining test concentrations of 0.10, 1.0 and 10% v/v saturated solution. The test item used represents a solution containing 29.2% anhydrous zicronium dichloride oxide. Given the low aqueous solubility it was considered that a loading rate of 50 mg test item/L, which corresponds to 14.6 mg zirconium dichloride oxide/L, was sufficient to ensure 100% saturation of the test media.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea, Daphnia magna
- Age at study initiation (mean and range, SD): < 24 h old (first instar)
- Strain: not reported, in-house laboratory culture

ACCLIMATION
- Acclimation conditions (same as test or not): Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Feeding frequency:daily
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
20-21°C
pH:
24 hours: 7.7-7.8
48 hours: 8.1
Dissolved oxygen:
air-saturation level
Salinity:
not applicable
Nominal and measured concentrations:
Nominal:
Range-finder: 0.10, 1.0, 10 and 100 % v/v saturated solution.
Measured: 100 % v/v saturated solution: < LOQ (0.051 mg Zr/L)
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL test and control vessel contained 200 mL of test media
- Aeration:no
- Renewal rate of test solution (frequency/flow rate): no renewal rate
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted from 6.8 to 7.8
- Photoperiod: 16 hours light, 8 hours dark with 20 minutes dawn and dusk transition period

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): after 24 hours and 48 hours the number of immobilized Daphnia magna were recorded

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: spacing factor was 10 as test was range finder study (no definitive study performed as no effects were seen in 100% v/v saturated solution)
- Range finding study: yes
- Test concentrations: 0.1-1.0-10 and 100% v/v saturated solutions
- Results used to determine the conditions for the definitive study: No immobility was observed at a loading of 100% v/v saturated solution, therefore, and because dissolved Zr levels were < LOQ in all treatments, no definitive study was performed.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: no measurable dissolved zirconium present in the range finder study
Details on results:
No mortality was observed in the preliminary test up to the highest test dose (100% v/v saturated solution).
Results with reference substance (positive control):
EC50 after 24 hours: 0.75 mg/L (CI: 0.56-1.0)
EC50 after 48 hours: 0.45 mg/L (CI: 0.42-0.48)
NOEC after 24 hours and 48 hours: 0.56 and 0.32 mg/L
Results of positive control with potassium dichromate were within the normal range for this reference item.

Chemical analysis of the test preparations at 0 and 48 hours showed measured dissolved zirconium concentrations of less than the limit of quantitation (LOQ) of the analytical method employed (which was determined to be 0.051 mg Zr/L). As a result, it was considered unnecessary to carry on to the definitive stage of testing.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item (zirconium dichloride oxide) to the freshwater invertebrate Daphnia magna has been investigated and yielded a 48-h EC50 value of greater than 100% v/v saturated solution. No mortality was observed up to and including at the highest test dose. No measurable dissolved zirconium was present in any of the test solutions (dissolved Zr < LOQ = 51 µg Zr/L). Therefore, no definitive toxicity test was conducted for zirconium dichloride oxide.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No information concerning the concentration used to determine the EC50 (24, 48 and 96 h) but results are based on a logarithmic scale as outlined in standard methods. No analytics were performed. The nominal concentrations used for this test were based on the results of preliminary acute static bioassays.
Qualifier:
according to guideline
Guideline:
other: APHA, AWWA, WPCF (1981) Standard methods for the examination of water and waste waters. American Public Health Association, 14th ed., Washington, DC
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
- Concentrations: A concentration gradient series from each respective stock solution was prepared in tubewell water. Final concentrations were based on the results of preliminary acute static bioassays. Test concentrations were selected on a logarythmic scale as outlined in standrad methods (APHA et al. , 1981).
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were prepared in distilled water.
Test organisms (species):
other aquatic worm: Tubifex tubifex
Details on test organisms:
TEST ORGANISM
- Common name: Tubifex tubifex
- Source: Tubificid worms were collected from Gheru Campus of ITRC, Lucknow, from natural sources.

ACCLIMATION
- Acclimation period: 7 days prior to experiment
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No post exposure observation period
Hardness:
245 mg/L as CaCO3
Test temperature:
30°C
pH:
7.6
Dissolved oxygen:
5.8 mg/L
Salinity:
Not relevant
Nominal and measured concentrations:
No data
Details on test conditions:
TEST SYSTEM
- Test vessel: 200 mL beakers containing 100 mL of test water.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- Renewal rate of test solution (frequency/flow rate): Test water was renewed every 24 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Test worms were considered dead when there was complete immobilization and no response to pression with a blunt glass rod.
The dead specimens were removed and recorded at intervals of 30 min and 1, 2, 4, 8, 14+/-2, 24, 33+/-3, 48 and 96 hours.

TEST CONCENTRATIONS
- Test concentrations: Test concentrations were selected on a logarithmic scale as outlined in standard methods (APHA et al. , 1981).
- Results of preliminary tests used to determine the conditions of the definitive study.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
403.3 mg/L
Conc. based on:
element
Remarks:
Zr
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 380.41-440.41 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
331.81 mg/L
Conc. based on:
element
Remarks:
Zr
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 320.06-366.24 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
221.18 mg/L
Conc. based on:
element
Remarks:
Zr
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 190.61-231.27 mg/L
Details on results:
In control tests, tubificid worms remained active during the test period. They were clustered at the bottom of the test container and showed typical tubificid movement.
Reported statistics and error estimates:
EC50 (effective concentration at wich 50% immobilization response was recorded) values and 95% confidence limits were calculated by the moving average angle method (Harris EK, 1959, Confidence limits for LD50 using the moving average angle method. Biometrics 15: 424-432).
Validity criteria fulfilled:
yes
Conclusions:
Zirconium dichloride oxide had no acute toxic effect on Tubifex tubifex after 96 h of exposure with the EC50 > 100 mg/L expressed as elemental zirconium or zirconium dichloride oxide.
Executive summary:

The acute toxicity of zirconium dichloride oxide to Tubifex tubifex was studied under semi-static conditions according to the APHA method. Tubifex tubifex were exposed to control and test chemical following a logarithmic scale for 96 hours. Mortality and immobilization were recorded at intervals of 30 min and 1, 2, 4, 8, 14, 33, 48 and 96 hours. No significant immobilization was observed after 96 hours exposure.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Read across from a study performed with zirconium dioxide. The read across justification document is attached to IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
anhydrous zirconium dichloride oxide
Basis for effect:
mobility
Remarks on result:
other: Based on the study performed with zirconium dioxide, it can be concluded that no immobilisation is to be expected at the limit test concentration (nominal loading rate of 100 mg/L, anhydrous zirconium dichloride oxide).

Description of key information

The endpoint was covered using a weight of evidence approach including two studies with zirconium dichloride oxide (Harris, 2014; Khangarot, 1991) and a read across study with zirconium dioxide (Bazin, 1994). All studies indicate that zirconium is not toxic to aquatic invertebrates, with the EC50 values being > 100% v/v saturated solution (Harris, 2014) or > 100 mg/L (initial loading rate, Khangarot, 1991; Bazin, 1994). Although zirconium dichloride oxide is a 'water soluble' compound, zirconium precipitates out of solution at environmentally relevant conditions, justifying read across from zirconium dioxide (which is an insoluble zirconium compound) to zirconium dichloride oxide.

Key value for chemical safety assessment

Additional information

Three relevant studies were identified and were used in a weight of evidence approach to cover this endpoint.

 

In the most recent study of Harris (2014), a range finding study was performed in which the highest test concentration was a 100% v/v saturated solution of zirconium dichloride oxide. No immobility was observed after 48 hours, which leads to an EC50 > 100% v/v saturated solution. Dissolved zirconium concentration was < LOQ (i.e. 0.051 mg Zr/L) in all treatments. Because zirconium was not available to cause effects in Daphnia magna, no further testing was performed.

The second study (Khangarot, 1991) investigated the toxicity of zirconium dichloride oxide to Tubifex tubifex. The worms were exposed up to 96 h. The EC50 values determined at 24 h, 48 h and 96 h were all > 100 mg/L expressed as elemental zirconium or zirconium dichloride oxide. The study was however considered reliable with restrictions since no analytics were performed and not all details on the results were shown.

Because of the precipitation of zirconium at environmentally relevant conditions, it is considered justified to perform read across from insoluble zirconium substances such as zirconium dioxide (for further argumentation reference can be made to the read across document attached to IUCLID Section 13). Therefore, the study from Bazin (1994) performed with zirconium dioxide (an insoluble zirconium compound) was included in the weight of evidence approach.

In this study, the acute toxicity of zirconium dioxide to Daphnia magna was studied under static conditions according to EU method C2. No significant immobilization was observed at a loading rate of 100 mg/L (ZrO2). The 48-h EC50 was thus superior to this value.

In conclusion, at environmentally relevant pH values, zirconium dichloride oxide can be concluded not to cause any acute adverse effects in aquatic invertebrates.