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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 05 August 2014 and 13 August 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Nominal and measured concentrations:
Nominal: 10 and 100 mg/L
Details on test conditions:
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.

Previous studies conducted on similar test items (e.g. Harlan Study Number 41304080) indicated that a 23-Hour stirring period followed by a 1-Hour standing period was sufficient to ensure that the maximum dissolved test item concentration was obtained in a Water Accommodated Fraction (WAF), as no significant increase in dissolved carbon was obtained by extending the stirring period to 96 hours.


Definitive Test
The following loading rates were assigned to the definitive test: 10 and 100 mg/L.


Experimental Preparation
Nominal amounts of test item (23 and 230 mg) were each separately added to the surface of
2.3 litres of test water to give the 10 and 100 mg/L loading rates respectively. Due to the volatile nature of the test item, all stirring vessels were completely filled and sealed. 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 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 approximate 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs.

Total Organic Carbon (TOC) analysis was performed on the test solutions at 0 and 48 hours.


Exposure Conditions
In the definitive test completely filled 100 mL nominal volume conical flasks containing approximately 130 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then sealed to reduce loss through volitilization and maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 16 hours light (618 to 645 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period.

Control data was shared with similar concurrent studies.


Evaluations
Test Organism Observations
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 other: mg/l loading rate Water-accommodated fraction
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: The toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole.
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 other: mg/l loading rate Water-accommodated fraction
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Total Organic Carbon Analysis
Total Organic Carbon (TOC) analysis of the test preparations at 0 and 48 hours showed no significant differences in the amount of carbon present within the 10 and 100 mg/L loading rate WAF test vessels when compared to the control vessels. Therefore, given the low background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered 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.


Immobilization Data
There was no immobilization in 20 daphnids exposed to concentrations of 10 and 100 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

The 24 h EL50 was > 100 mg/L Loading Rate WAF
The 48 h EL50 was > 100 mg/L Loading Rate WAF

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF.
Results with reference substance (positive control):
A positive control (Harlan Study Number 41400711) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the maximum-likelihood probit method (Finney, 1971 ) at 24 hours and by the trimmed Spearman-Karber method (Hamilton et al 1977**) at 48 hours based on the nominal test concentrations gave the following results:
The 24 h EC50 was 0.87 mg/L with 95 % confidence limits of 0.75 – 1.0 mg/L.
The 48 h EC50 was 0.71 mg/L with 95 % confidence limits of 0.65 – 0.78 mg/L.
The NOEC was 0.56 mg/L and the LOEC was 1.0 mg/L.

The No Observed Effect Concentration is based upon less than 10% immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
An estimate of the EL50 values was given by inspection of the immobilization data.

Validation Criteria

The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

 

Water Quality Criteria

Temperature was maintained at approximately 20°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 the 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 10 and 100 mg/L loading rates were observed to be clear colorless water columns with globules of test item at the surface. After 23 hours stirring and a
1-Hour standing period the 10 and 100 mg/L loading rates were observed to be clear colorless water columns with a slick of 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 10 and 100 mg/L loading rates were observed to be clear, colorless solutions. 

Validity criteria fulfilled:
yes
Conclusions:
Measured acute (short-term) toxicity data are available for Shell GTL Solvent GS270 (Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics) to the freshwater invertebrate Daphnia magna. The test was conducted under static (no renewal of the test media) conditions in accordance with OECD Test Guideline 202 and method C2 of EC Regulation No. EC 440/2008. Appropriate modifications to the test and media preparation procedures were made to take account of the test substance containing multiple constituents, having low solubility in water and being potentially volatile. No effects on mobility of D. magna were observed after 48 hours exposure to the test medium prepared as a water-accommodated fraction (WAF) at loading rates up to 100 mg/l; 48-hour EL50 >100 mg/l, NOELR ≥100 mg/l. Samples of the control and the 10 and 100 mg/l loading rate WAFs were taken at 0 and 48 hours for Total Organic Carbon (TOC) analysis. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method. The results of the test are considered to be reliable.
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 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 the definitive test, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item at nominal loading rates of 10 and 100 mg/L for 48 hours at a temperature of approximately 20°C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Results….

Samples of the control and the 10 and 100 mg/L loading rate WAFs were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method.

 

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 Daphnia magna to the test item gave EL50values 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 test substance is poorly soluble and made of constituents with various water solubility. As a consequence, the exposures were performed with Water Accommodated Fractions (WAFs). Therefore, the results are based on nominal loadings and the LC50 value given below is actually a LL50 (lethal loading) value. No LC50 could be derived from the acute toxicity studies. The 48h-LL50 is > 100 mg/l.

Key value for chemical safety assessment

Additional information

Measured acute (short-term) toxicity data are available for Shell GTL Solvent GS270 (Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics) to the freshwater invertebrate Daphnia magna (Harlan Laboratories, 2014). The test was conducted under static (no renewal of the test media) conditions in accordance with OECD Test Guideline 202 and method C2 of EC Regulation No. EC 440/2008. Appropriate modifications to the test and media preparation procedures were made to take account of the test substance containing multiple constituents, having low solubility in water and being potentially volatile. No effects on mobility of D. magna were observed after 48 hours exposure to the test medium prepared as a water-accommodated fraction (WAF) at loading rates up to 100 mg/l; 48-hour EL50>100 mg/l, NOELR ≥100 mg/l. Samples of the control and the 10 and 100 mg/l loading rate WAFs were taken at 0 and 48 hours for Total Organic Carbon (TOC) analysis. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method. The results of the test are considered to be reliable.