Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]

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:

22 July 2017 - 22 August 2017

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:

yes

Vehicle:

not specified

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 24 hours and the mixture allowed to stand for 24 hours. 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 WAF 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 100 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48, 72 and 96 hours

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 Water Accommodated Fraction (WAF) of the test item. At the request of the Sponsor the test item was prepared using a 24-Hour stirring period followed by a 24-Hour standing period prior to removal of the aqueous phase for testing.

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 31 May 2017. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 17 July 2017 to 24 July 2017. 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 15 °C to 16 °C with a dissolved oxygen content of greater than or equal to 9.0 mg O2/L. These parameters were recorded daily.

The stock fish were fed commercial trout pellets which was discontinued approximately 23 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 5.3 cm (sd = 0.34) and a mean weight of 1.23 g (sd = 0.25) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.43 g bodyweight/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:

static

Water media type:

not specified

Limit test:

yes

Total exposure duration:

96 h

Hardness:

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.

Test temperature:

15 °C to 16 °C

pH:

7.66

Nominal and measured concentrations:

Nominal loading rate of 100 mg/L.
Analysis of the freshly prepared test preparations at 0, 24, 48 and 72 hours showed that measured concentrations of between 0.024 and 0.73 mg/L were obtained.

Details on test conditions:

Definitive Test
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 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.

Experimental Preparation
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 24 hours and the mixture allowed to stand for 24 hours. 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 WAF 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 100 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48, 72 and 96 hours

Exposure Conditions
In the definitive test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. 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 15 °C to 16 °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 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.

Reference substance (positive control):

yes

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Details on results:

Analysis of the freshly prepared media (see Annex 2) at 0, 24, 48 and 72 hours showed that measured concentrations of between 0.024 and 0.73 mg/L were obtained. Analysis of the old or expired test preparations at 24, 48, 72 and 96 hours showed that measured concentrations of between 0.013 and 0.44 mg/L were obtained.
Analysis of the control samples from the 0 and 72 hour fresh media and the 24 and 96 hour old media showed that measured concentrations of test item were observed to be present. Given that no toxicity was observed throughout the duration of the test, this was considered not to have an an impact on the outcome or integrity of the test. Control samples taken at 24 and 48 hours (fresh media) and 48 and 72 hours (old media) showed measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method employed were obtained, determined to be 0.0077 mg/L. These samples were analyzed on subsequent days to the corresponding test samples suggesting that the measured concentrations observed in the initial control analysis may have been due to post sampling contamination.
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.

There were no mortalities in 7 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 7 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 (6.1 mg O2/L) in the control and test vessels.

Temperature was maintained at 15 to 16 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

At the start of each mixing period the 100 mg/L loading rate was observed to be a clear colorless water column with an oily layer or globules of test item on the surface. After
24 hours stirring and a 24-Hour standing period the 100 mg/L loading rate of the 0 and 24-hour preparations were observed to be clear colorless water columns with globules of test item floating on the surface and settled on the bottom. The 48 and 72-Hour preparations were observed to be clear colorless water columns with an oily layer or globules of test item on the surface. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the control and the 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 No Observed Effect Loading 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 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 toDaphnia magnastudy 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 toDaphnia magnastudy 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 Water Accommodated Fraction (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 15°C to 16 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

Analysis of the freshly prepared test preparations at 0, 24, 48 and 72 hours showed that measured concentrations of between 0.024 and 0.73 mg/L were obtained. Analysis of the old or expired test preparations at 24, 48, 72 and 96 hours showed that measured concentrations of between 0.013 and 0.44 mg/L were obtained.  

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 than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 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 LL₅₀value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading 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