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:
Experimental start date 30 July 2017 Experimental completion date 12 September 2017
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)
Specific details on test material used for the study:
Identification: 3,6,9-tris(ethyl and propyl derivatives) 3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane
Description/Appearance: Clear, colourless liquid
Batch Number: BYK004856-98 (final)
Purity: Not applicable, UVCB
Expiry Date : 03 January 2024
Storage Conditions: Ambient, in darkness
Analytical monitoring:
yes
Details on sampling:
Chemical Analysis of Test Loading Rates
Water samples were taken from the control and the 100 mg/L loading rate WAF test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.

Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Experimental Design and Study Conduct
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 using a 23-Hour stirring period flowed by a 1-Hour settlement period.

Definitive Test
Based on data provided by the Sponsor, a "Limit test" was conducted at a single loading rate of 100 mg/L to confirm that no mortalities or sub-lethal effects of exposure were observed.

Experimental Preparation
A nominal amount of test item (2250 mg) was added to the surface of 22.5 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. 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 and 72 hours (fresh media) and at 24, 48 and 96 hours (old media).
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 tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 24 July 2017 to 31 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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
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. On the first day of testing, the hardness was measured to be 104 mg/L as CaCO3, however, given that no adverse effects were observed on the test organisms, this was considered not to have had an impact on the outcome of the test.
Test temperature:
Water temperature was controlled at 15°C to 16°C
pH:
7.3 to 7.6
Dissolved oxygen:
9.5 to 9.9 mg O2/L
Salinity:
Not applicable as freshwater study
Conductivity:
345 μS/cm at 20°C
Nominal and measured concentrations:
Based on data provided by the Sponsor, a "Limit test" was conducted at a single loading rate of 100 mg/L to confirm that no mortalities or sub-lethal effects of exposure were observed.
Details on test conditions:
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 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.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Please refer to "Any other information on results incl. tables"
Results with reference substance (positive control):
Not applicable

 DefinitiveTest

 Chemical Analysis of Test Loading Rates

Analysis of the freshly prepared 100 mg/L test preparation at 0, 24 and 72 hours showed that measured concentrations of between 2.3 and 4.7 mg/L were obtained. Analysis of the old or expired media at 24, 48 and 96 hours showed that measured concentrations of between less than the Limit of Quantification (LOQ), of the analytical method, determined to be 0.40 mg/L, and 2.8 mg/L were obtained.

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 7 fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h)

LL50

(mg/L Loading Rate WAF)

3

>100

6

>100

24

>100

48

>100

72

>100

96

>100

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

Sub-Lethal Effects

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.

 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 15 °C to 16 °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 WAF.

At the start of the mixing period the 100 mg/L loading rate was observed to be a clear colorless water column with an oily layer of test item on the surface. 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 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 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 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.

It was considered unnecessary and unrealistic to test at loading rates in excess of 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).

Based on data provided by the Sponsor, 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 100 mg/L test preparation at 0, 24 and 72 hours showed that measured concentrations of between 2.3 and 4.7 mg/L were obtained. Analysis of the old or expired media at 24, 48 and 96 showed that measured concentrations of between less than the Limit of Quantification (LOQ), of the analytical method, determined to be 0.40 mg/L, and 2.8 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.

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

Description of key information

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss) according to OECD 203 under GLP.

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). A semi-static limit test at 100 mg/L loading was performed. 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.

One OECD 203 GLP study is available with one of the main components.  The EC50 is greater than the maximum water solubility, thus it is not acutely toxic to fish.  One OECD 236 study is available, the  EL50 > 100 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

OECD 203 (CAS 24748 -23 -0)

The test concentration used (2.0 mg/L nominal concentration) was the highest attainable concentration which could be achieved. A discrepancy with the water solubility test as reported in this dossier is noted. In the preliminary water solubility test, however, solubility up to 2 mg/L was found and this value was used for the fish toxicity study. More vigorous testing conditions in the final water solubility study led to a slightly higher solubility level. Analysis of the freshly prepared solutions were in excess of 80% nominal test concentrations. Analysis at the other sampling times showed a decrease in concentration which is assumed to be attributed to the poor water solubility and aeration of the test vessels which may have caused phase separation of the test media.

OECD 236 (CAS 1613243 -54 -1)

Test concentrations and controls

WAF preparations of 1, 10 and 100 mg/L including a control in test medium were prepared. In addition a positive control of 3,4-dichloro aniline at a concentration of 4 mg/L was prepared as indicated by the test guideline. Due to the positive control being prepared with acetone, a solvent control was also required according to the test guideline. A solvent control of 100µl/L was therefore also prepared.

The 96 hour raw data only has been reported as only this is required for the primary endpoint, Lethality after 96 hours. 

 

NOELR = 100 mg/L

EL50= > 100 mg/L

No significant lethal effects were therefore observed by the test substance and the test plate can be considered valid based on the results of the internal plate control exceeding 80% survival.

 

The test substance at its solubility limit in test medium can be concluded to cause no lethal toxic effects to zebra fish embryos. Due to no effects being observed at the highest loading rate no statistical analysis is required.