Registration Dossier

Ecotoxicological information

Short-term toxicity to fish

Currently viewing:

Administrative data

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:
21 March 2018 - 16 April 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:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not specified
Details on sampling:
Water samples were taken from the control and 100 mg/L loading rate WAF test group 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 72 (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:
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 there to be no micro-dispersions of test item present. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 72 and 96 hours

The test item concentration in the test samples was determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard. The test item gave a chromatographic profile consisting of a single peak.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
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 09 March 2018. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 15 March 2018 to 22 March 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 10.0 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 19 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.1 cm (standard deviation = 0.14) and a mean weight of 0.5 g (standard deviation = 0.11) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.175 g body weight/liter.
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
Test temperature:
14°C to 15 °C
pH:
7.0
Dissolved oxygen:
10.9
Nominal and measured concentrations:
Analysis of the freshly prepared test preparations at 0 and 72 hours (see Annex 4) showed that measured concentrations of 0.93 and 11 mg/L respectively were obtained. Analysis of the old or expired test preparations at 24 and 96 hours showed that measured concentrations of 1.0 and 20 mg/L were obtained respectively.
Details on test conditions:
In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. 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 KY89WQ and YG67TK 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.

For the control and test concentration, 25 to 30 liter glass exposure vessels containing 20 liters of test media were used. At the start of the test 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 at approximately 14 C to 15 C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. 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.
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
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.

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.
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.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAF.
At the start of the mixing period the 100 mg/L loading rate was observed to be clear colorless water column with oily globules/oily layer of test item floating on the surface and dispersed throughout the column. After 23 hours stirring and a 1 Hour standing period the 100 mg/L loading rate was observed to remain as at the start of stirring. Microscopic examination of the WAF showed microdispersions present and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all test item/microdispersions.
During the test the control and 100 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 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.

1.1               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 recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EL50value 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 LL50is greater than the threshold loading rate. Therefore, as the EL50value obtained for both the Algal Growth Inhibition study and the Acute Toxicity to Daphnia magna study (Envigo study numbersKY89WQ and YG67TKrespectively) 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 °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 1, 3 and 5 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

1.1               Results

Analysis of the freshly prepared test preparations at 0 and 72 hours showed that measured concentrations of 0.93 and 11 mg/L respectively were obtained. Analysis of the old or expired test preparations at 24 and 96 hours showed that measured concentrations of 1.0 and 20 mg/L were obtained respectively.

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 LL50values of greater than100 mg/L loading rate WAF. The No Observed Effect Loading (NOEL) Rate was100 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

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.

Key value for chemical safety assessment

Additional information