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Ecotoxicological information

Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-23 - 2017-05-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- Sampling method: The test samples were thawed with the aid of sonication, filtered through a 0.45 μm cellulose acetate filter, and then analysed.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test item for the purposes of testing the test item was prepared as a Water Accommodated Fraction (WAF). A study to determine the General Physico-Chemical Properties of the test item indicated that the water solubility of the test item was less than 1.0 mg C/L. given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading the aqueous phase with undissolved test item. A 23-Hour stirring period, followed by a 1-Hour standing period was deemed sufficient to ensure equilibration between the test item and aqueous phase.

A nominal amount of test item (1100 mg) was added to the surface of 22 liters of test water to give the 50 mg/L loading rate. A nominal amount of mineral oil (583 mg) was added to the surface of 22 liters of test water to give a nominal loading rate of 26.5 mg/L. After the addition of the test item and mineral oil, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the mineral oil control and the 50 mg/L loading rate WAF.

The concentration of boron in the test preparations were verified by chemical analysis at 0, 24, 27 and 96 hours

Total Organic Carbon (TOC) analysis was performed on the test solutions at 0, 24, 72 and 96 hours
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Length at study initiation (length definition, mean, range and SD): 5.5 cm (sd = 0.4)
- Weight at study initiation (mean and range, SD): 1.33 g (sd = 0.31)

ACCLIMATION
- Acclimation period: 2017-04-17 - 2017-04-23.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food during acclimation: commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test.
- Health during acclimation (any mortality observed): no mortalities

FEEDING DURING TEST
- none
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
none
Hardness:
approximately 140 mg/L as CaCO3
Test temperature:
14 °C to 15 °C
pH:
7.5 - 8.0
Dissolved oxygen:
≥ 9.5 mg O2/L.
Salinity:
not applicable
Conductivity:
392.342 μS/cm at 20°C
Nominal and measured concentrations:
definite test: 50 mg/L loading rate WAF
Details on test conditions:
TEST SYSTEM
- Test vessel: 20 L glass exposure vessels
- Type (delete if not applicable): closed (vessels were covered)
- Material, size, headspace, fill volume: glass
- Aeration: aerated via narrow bore glass tubes
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 7
- No. of vessels per control (replicates): 1 (definitive test)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener. Dechlorinated.
- Total organic carbon: 1.060 mg/L
- Pesticides: < 0.01 µg/L (used tap water)
- Chlorine: 0.333 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test with 50 mg/L)

Reference substance (positive control):
no
Remarks:
Not mandatory under the applied guideline.
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
other: mortality and sub-lethal effects
Details on results:
- Mortality of control: none
- Other adverse effects control: no
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
No statistics were reported.

Definitive Test

Chemical Analysis of Test Loading Rates

Analysis of the freshly prepared test preparations at 0 and 72 hours showed that measured concentrations of 0.049 and 0.026 mg/L as boron were obtained (equivalent to 1.3 and 0.67 mg/L as test item). In the old or expired media at 48 and 96 hours measured concentrations of 0.049 and 0.026 mg/L as boron was obtained (equivalent to 1.3 and 0.70 mg/L as test item). Concentrations of less than the limit of quantification of the analytical method, determined to be 0.020 mg/L as boron (equivalent to 0.53 mg/L as test item), were obtained in the control and mineral oil control at 0 and 72 hours.

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Total Organic Carbon Analysis

Total Organic Carbon (TOC) analysis of the test preparations at 0, and 72 hours (fresh media) and at 24 and 96 hours (old media) showed that all the results were around the limit of quantification of the analytical method.

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Mortality Data

There were no mortalities in seven fish exposed to a 50 mg/L loading rate WAF for a overall period of 96 hours and assessed at 3, 6, 24, 48, 72 and 96h. The No Observed Effect Loading rate (NOEL) was determined to be 50 mg/L loading rate WAF.

Sub-Lethal Effects

There were no sub-lethal effects of exposure observed in seven fish exposed to a 50 mg/L loading rate WAF for a period of 96 hours.

Validation Criteria

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV in the control and test vessels.

Water Quality Criteria

Temperature was maintained at 14 °C to 15 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Vortex Depth Measurements

The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAFs.

At the start of the mixing period the mineral oil control was observed to be a clear colorless water column with mineral oil test item at the surface. At the start of the first, third and forth mixing periods, the 50 mg/L loading rate was observed to be a clear colorless water column with test item on the surface and on the base of the vessel, and at the start of the second mixing period was observed to be a clear colorless water column with test item on the surface. After 23 hours stirring and a 1-Hour standing period the mineral oil control was observed to be a clear colorless water column with mineral oil test item at the surface. After the first, second and fourth stirring and a 1-Hour standing period, the 50 mg/L loading rate was observed to be a clear colorless water column with test item at the surface and at the base of the vessel, whilst after the third stirring and standing period, the 50 mg/L loading rate was observed to be a clear colorless water column with test item at the surface and adhered to the glass tube and some white and waxy test item on the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the control, mineral oil control and the 50 mg/L loading rate were observed to be clear, colorless solutions.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated using the threshold approach and gave a 96-Hour LL50 value of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading rate was 50 mg/L loading rate WAF.
Executive summary:

A GLP study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility of < 1.0 mg C/L and complex nature of the test item for the purposes of testing the test item was prepared as a Water Accommodated Fraction (WAF) with a maximum of 50 mg/L to prevent overloading the aqueous phase with undissolved test item. Since the threshold of 50 mg/L loading rate WAF was the lowest EL50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study, thie study has been conducted as "Limit test" with 50 mg/L loading rate WAF.

Seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 50 mg/L for a period of 96 hours at a temperature of 14 °C to 15 ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of rainbow trout to the test item gave LL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF.

Description of key information

Acute toxicity to freshwater fish, OECD 203, semi-static, freshwater, Oncorhynchus mykiss: LL50(96h) > 50 mg/L WAF

Key value for chemical safety assessment

LC50 for freshwater fish:
50 mg/L

Additional information

In the GLP key study (Ablitt, 2017b) the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss) was assessed via the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility of < 1.0 mg C/L and complex nature of the test item for the purposes of testing the test item was prepared as a Water Accommodated Fraction (WAF) with a maximum of 50 mg/L to prevent overloading the aqueous phase with undissolved test item. Since the threshold of 50 mg/L loading rate WAF was the lowest EL50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study, the study has been conducted as "Limit test" with 50 mg/L loading rate WAF.

Seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 50 mg/L for a period of 96 hours at a temperature of 14 °C to 15 ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of rainbow trout to the test item gave LL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF.