Molybdenum trioxide, reaction products with bis[O,O-bis(2-ethylhexyl)] hydrogen dithiophosphate

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:

05 April 2018 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

-Purity: Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
-Description: Dark Green Liquid

Analytical monitoring:

yes

Remarks:

Given that the test substance is a multi-constituent substance and analytical monitoring was limited to the dithiophosphate fraction (without molybdenum) of the test substance, the results were based on nominal loading rates.

Details on sampling:

- Concentrations: Control and 100 mg/L loading rate WAF
- Sampling method: Aqueous samples (10 mL) of freshly prepared test media at 0 (fresh media), 24 (old media), 48 (fresh media) and 72 hours (old media) were collected in duplicate, mixed with acetonitrile, then diluted further with test water:acetonitrile (1:1, v/v) as required to bring the response within the calibration range.
- Sample storage conditions before analysis: Not applicable

Vehicle:

no

Details on test solutions:

The substance is a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) with low water solubility and as such falls into the category of a “difficult substance” as defined by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). Based on the recommendations of this guideline, the test substance was prepared as Water Accommodated Fractions (WAFs). At the start of the test, and each media renewal, test substance was weighed onto a glass slide, placed in the test media. The preparation was stirred for ca 23 hours at a rate such that a slight dimple was formed at the surface, then allowed to settle for ca 1 hour after which the aqueous phase was removed by careful mid-depth siphoning to give the 100 mg/L loading rate WAF. The control solution was prepared in the same manner without the addition of test substance.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Northern Trout (United Kingdom)
- Length at study initiation): 5.1 cm (mean); 4.8 - 5.5 cm
- Weight at study initiation: 1.2 g (mean); 0.9 - 1.5 g

ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions: same as test conditions
- Type and amount of food during acclimation: Fish were fed during acclimation with a proprietary fish food, which was added to the holding tank in quantities dictated by the size of the fish. Uneaten food and debris was siphoned or cleaned from the tanks as required
- Health during acclimation (any mortality observed): 0.1% mortality observed in the stock batch of fish in the seven days prior to test initiation.

FEEDING DURING TEST
The fish were not fed during the 24 hours before the start of the definitive test and were not fed throughout the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

18.4 mg/L as CaCO3 (typical)

Test temperature:

14.7 - 16.6ºC

pH:

6.95 - 7.65

Dissolved oxygen:

93 - 104% air saturated value (ASV)

Salinity:

Not applicable

Conductivity:

183 uS/cm (typical)

Nominal and measured concentrations:

0 (control) and 100 mg/L loading rate WAF (nominal).
All measured concentrations were based on quantitation of dithiophosphate fraction (without molybdenum) and were below the limit of quantification (1 mg/L).

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 L constructed glass aquaria, each fitted with a clear plastic lid
- Volume of test media: 15 L
- Aeration: yes; sufficient to maintain oxygen concentration
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 0.54 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Treated tap water; passed through activated carbon filters
- Alkalinity: 18.4 mg/L (as CaCO3)
- Ca/mg ratio: 6
- Culture medium different from test medium: same
- Intervals of water quality measurement: bi-annually

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 hour light: 8 hours dark lighting cycle with a 30 minute transition period
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Fish in each test vessel were observed for mortality and sublethal effects at ca 3 hours and at 24-hour intervals (24, 48, 72 and 96 hours) throughout the duration of the test.

TEST CONCENTRATIONS
The definitive test was conducted at a single loading rate of 100 mg/L according to the threshold concept (OECD Series on Testing and Assessment No. 126, 2010). The selected loading rate was based on the results from the algal growth inhibition and acute Daphnia studies conducted under static conditions with the same test substance.

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: mg/L loading rate WAF

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: mg/L loading rate WAF

Basis for effect:

mortality (fish)

Details on results:

- Behavioral abnormalities: none
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations that might cause a difference between measured and nominal values: The test preparations were observed to be colourless solutions throughout the duration of the test. The test substance was a challenging analytical target due to the complex nature of the compound. The loss of sensitivity observed across an analytical run, which greatly impacted instrument performance, was attributed to a build-up of metal ions onto the ion optics in the instrument, where their presence changed the charging and electronic characteristics of the quadrupole rods and ion lenses, in such a way that the instrument was not able to self-correct within a run. This behavior seemed to worsen over time and/or in select media used in the different studies with this test substance. The test substance was also found to have extremely limited stability in any of the solvents suitable for LC/MS analysis, making repeat analysis or even standard inter-comparison unviable.
- Effect concentrations exceeding solubility of substance in test medium: yes

Reported statistics and error estimates:

Statistical analysis was not performed given that no mortality was observed during the test. The LL50 values, no observed effect loading rate (NOEL) and the lowest observed effect loading rate (LOEL) were estimated empirically by observation of the data.

Sublethal observations / clinical signs:

Mortality of fish recorded during the definitive test

Nominal loading	Cumulative number of mortalities recorded*
rate (mg/L)	24 hours	48 hours	72 hours	96 hours
Control	0	0	0	0
100	0	0	0	0
* Seven fish added to each test vessel at test initiation.

Validity criteria fulfilled:

yes

Remarks:

The validity criteria for control mortality (less than 10%, actual = 0%) and dissolved oxygen (>60 % ASV, actual ≥93%) were both satisfied. The test was therefore considered to be valid.

Conclusions:

Based on nominal loading rates, the 96-hour LL50 value was estimated to be >100 mg/L. The NOEL was considered to be 100 mg/L. The test showed no toxicity at the limit of solubility of the test substance in the test media.

Executive summary:

The study was conducted in accordance with the requirements of OECD Chemicals Testing Guideline No. 203 to assess the toxicity the 96-hour acute toxicity of the substance to rainbow trout (Oncorhynchus mykiss). Test solutions were prepared as Water Accommodated Fractions (WAFs) based on the recommendations of OECD Series on Testing and Assessment, No. 23. 

The definitive test was conducted as a ‘Limit Test’ under semi-static conditions (daily renewal) at a single nominal loading rate of 100 mg/L with a concurrent control group, each consisting of a single 20 L constructed glass aquaria containing 15 L of media. Following acclimation to the test conditions for at least 12 days, seven fish were added to each test vessel. TheDaphnia magna in each test vessel were observed for mortality and sub-lethal effects at ca. 3 hours and at 24-hour intervals (24, 48, 72 and 96 hours) throughout the duration of the test. The fish were not fed during the test.

Test solutions were analysed at 0 and 48 hours (fresh media) and at 24 and 72 hours (old media) using high performance liquid chromatography (HPLC) with time of flight mass spectrometry (TOF) based on quantitation of the dithiophosphate fraction (without molybdenum) of the test substance. The analytical method was successfully validated at concentrations of 1 and 10 mg/L however, the attempted validation at 0.1 mg/L failed. As such, the limit of quantification (LOQ) using this method was considered to be 1 mg/L. All measured concentrations were based on quantitation of dithiophosphate fraction (without molybdenum) and were below the limit of quantification (1 mg/L), but the measured concentrations confirmed that test substance was present in the test media and remained stable over a 24-hour period.

Given that the test substance is a multi-constituent substance and analytical monitoring was limited to the dithiophosphate fraction (without molybdenum) of the test substance, the effect concentrations were based on nominal loading rates.

Based on nominal loading rates, the 96-hour LL50 was determined to be >100 mg/L. The NOEL was determined to be 100 mg/L. The test showed no toxicity at the limit of solubility of the test substance in the test media.

Description of key information

In an OECD Guideline No. 203 study conducted according to GLP to assess the toxicity of the substance to the freshwater fish species, Oncorhynchus mykiss, the 96-hour LL50 was determined under semi-static test conditions to be greater than 100 mg/L loading rate WAF and the No Observed Effect Loading rate (NOEL) was determined to be 100 mg/L loading rate (Smithers Viscient (ESG) Ltd, ****).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L