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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:
Experimental start date: 14 January 2018; Experimental completion date: 13 February 2018
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:
yes
Remarks:
Please see "Principles of methid if other than guideline" for details.
Principles of method if other than guideline:
The following deviation from the study plan occurred:
The expiry date of test item received was different to that documented in the study plan. The correct expiry date was 01 October 2018. Given that all testing was completed before the expiry date, this deviation was considered not to have adversely affected the results or integrity of the study.
This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and 100 mg/L loading rate WAF test vessel at 0, 24, 48 and 72 hours from fresh media and at 24, 48, 72 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.
Duplicate samples at 0 and 24 hours (fresh media) and 24 and 96 hours (old media) were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A nominal amount of test item (2200 mg) was added to the surface of 22 liters of test water to give the 100 mg/L loading rate. After the addition of the test item, 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 mixture allowed to stand for 1-Hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed fine particles of test item to be present, therefore the WAF was filtered through two sheets of filter paper. Following filtration through two filter papers, no particles of test item were observed.
The vortex depth was recorded at the start and end of each mixing period.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 18 December 2017.
There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.8 cm (standard deviation = 0.3) and a mean weight of 0.9 g (standard deviation = 0.2) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.3 g body weight/liter.

ACCLIMATION
Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 08 January 2018 to 14 January 2018. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 9.8 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 27 hours prior to the start of the definitive test.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
140 mg/L as CaCO3
Test temperature:
14 - 15 °C
pH:
7.5 - 8.1
Dissolved oxygen:
Greater than or equal to 9.8 mg O2/L
Salinity:
n/a freshwater study
Conductivity:
345 μS/cm at 20 °C
Nominal and measured concentrations:
Nominal: 100 mg/L
Details on test conditions:
TEST SYSTEM
For the control and test item WAF concentration, 25 to 30 liter glass exposure vessels containing 20 liters of test media were used. At the start of the experiment seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 15 °C with a maximum deviation of ±1 °C. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products. - Test vessel:

TEST MEDIUM / WATER PARAMETERS
The test water used for the definitive test was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Annex 2 attached below.
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
The pH of the freshly prepared 100 mg/L loading rate WAF was outside of the range 6.0 to 9.0 on each occasion (actual measured pH were between 9.5 and 9.6), therefore the pH was adjusted by addition of 1.0 M hydrochloric acid to match the pH of the control media.
Photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.

EFFECT PARAMETERS MEASURED: Any mortalities and sub-lethal effects of exposure were recorded at 1, 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
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.
Definitive Test
In accordance with the requirements of REACh, the test was conducted according to the threshold approach recommended by ECHA (ECHA 2017). Using this approach, the lowest EL50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate. Therefore, as the EL50 value obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study (Envigo study numbers 41500865 and 41500864 respectively) were greater than 100 mg/L loading rate WAF, the test was conducted at a single loading rate of 100 mg/L loading rate WAF to ensure that toxicity was not observed at this loading rate.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72 and 96 hours.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
other: sub-lethal effects
Details on results:
There were no mortalities in seven fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours. The NOEL rate was 100 mg/L loading rate WAF.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.
There were no sub-lethal effects of exposure observed in seven fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.
Sublethal observations / clinical signs:

Validation of Mixing Period

Preliminary investigational work indicated that there was no increase in the measured concentrations of aluminum when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Chemical Analysis of Test Loading Rates

Chemical analysis of the freshly prepared 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured test aluminium concentrations of between 0.33 and 0.43 mg/L. Chemical analysis of the corresponding old test solutions at 24 and 96 hours showed measured test aluminium concentrations of between 0.31 and 0.42 mg/L.

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.

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 (6.1 mg O2/L) in the control and test vessels.

Water Quality Criteria

Temperature was maintained at 14 to 15 °C throughout the test. While there were no treatment related differences for oxygen concentration, the pH of the 100 mg/L loading rate WAF was increased by the presence of the test item and was therefore adjusted to match the pH of the control media by addition of 1.0 M hydrochloric acid.

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 100 mg/L loading rate was observed to be a clear colorless water column with test item floating at the surface, dispersed throughout and settled at the bottom of the vessel. After 23 hours stirring and a 1-Hour standing period the 100 mg/L loading rate was observed to remain a clear colorless water column with test item floating at the surface, dispersed throughout and settled at the bottom of the vessel. Examination of the WAF at the end of stirring showed a test item was present in the water column and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). Microscopic observations of the WAF were performed after filtering and showed fine particles of test item to be present, therefore the WAF was filtered using two filter papers. Following filtration through two filter papers, no particles of test item were observed. During the test the 100 mg/L loading rate was observed to be a clear, colorless solution.

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 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.
Executive summary:

Introduction

A 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.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

In accordance with the requirements of REACh, the test was conducted according to the threshold approach recommended by ECHA

(ECHA 2017). Using this approach the lowest EL50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate. Therefore, as the EL50 value obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study (Envigo study numbers 41500865 and 41500864 respectively) were greater than 100 mg/L loading rate WAF, the test was conducted at a single loading rate of 100 mg/L loading rate WAF to ensure that toxicity was not observed at this loading rate.

Seven fish were exposed to a WAF of the test item, at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature

of 14 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 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours (fresh media) showed measured test aluminium

concentrations of between 0.33 and 0.43 mg/L. Chemical analysis of the corresponding old test solutions at 24 and 96 hours showed measured test aluminium concentrations of between 0.31 and 0.42 mg/L.

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 100 mg/L loading rate WAF. The No Observed Effect Loading

(NOEL) Rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

Exposure of rainbow trout to the test item gave LL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading (NOEL) Rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

A 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.

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.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

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