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Ecotoxicological information

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 04 August 2014 and 08 August 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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 liters of test water to give the 10 and 100 mg/L loading rates respectively. 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 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 volatilization and maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 16 hours light (398 to 508 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.
Data from the control group 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 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Water-accommodated fraction
Remarks:
The toxicity cannot be attributed to a single constituent or a mixture of constituents, but to the test item as a whole.
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Water-accommodated fraction
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 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 significant 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

A single immobilized daphnid was observed in the 10 mg/L Loading rate WAF test group and two immobilized daphnids were observed in the 100 mg/L Loading rate WAF test group. However, given that no greater than 10% immobilization occurred, it was considered that this was due to natural causes and was considered not to be significant.

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 21°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 the 1-Hour settlement period, the 10 and 100 mg/L loading rates were observed to be clear colorless solutions. 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 Hydrocarbons, C18-C24, isoalkanes, <2% aromatics to the freshwater invertebrate Daphnia magna. The test was conducted under static conditions (no renewal of the test media) in accordance with OECD Test Guideline 202. 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 a loading rate of 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 toDaphnia 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 21°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 ofDaphnia magnato 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

(48-h) EL50 >100 mg/l D. magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

Measured acute (short-term) toxicity data are available for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics to the freshwater invertebrate Daphnia magna (Sacker, 2014). The test was conducted under static conditions (no renewal of the test media) in accordance with OECD Test Guideline 202. 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 a loading rate of 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.

The acute toxicity of a sample of GTL Gasoil has been determined by Palmer (2001) in a test with the freshwater invertebrate Daphnia magna (water flea). The test was conducted in accordance with OECD 202 methodology.

WAFs of the sample were prepared in sealed vessels with minimum headspace by stirring for approximately 72 hours. The contents of the vessels were left to stand for 1-2 hours before drawing off the aqueous phase – the WAF – for testing. Static exposures were then carried out in completely full, sealed vessels. The tests were not subject to GLP and the test media were not analysed for stability or for exposure concentration of the test substance. However in other respects they were considered to fulfil the requirements of current best practice.

The test results, expressed as the EL50 value, showed that the sample was not acutely toxic to Daphnia magna at a loading rate of 1000 mg/l.

Albertus and Phillips (2005) have reported results for an acute toxicity test carried out on a second sample of the substance with the freshwater invertebrate Daphnia magna (water flea). The test was conducted in accordance with OECD Test Guidelines 202. The test media were water-accommodated fractions of the test sample that were prepared in sealed vessels with a small headspace according to the CONCAWE methodology for the ecotoxicological testing of petroleum products (CONCAWE report, 1993).

The test results, expressed as the EL50 value, showed that the sample was not acutely toxic to Daphnia magna at a loading rate of 1000 mg/l.

The short-term toxicity of GTL Base Oil Distillates with the freshwater invertebrate Daphnia magna (water flea) has been determined by Harlan (Priestly, 2009) in a study conducted in accordance with OECD Test Guideline 202. 

WAFs of the sample were prepared by stirring for approximately 23 hours and left to stand for 1 hour before drawing off the aqueous phase – the WAF – for testing. Static exposures were then carried out according to GLP.

Total Organic Carbon (TOC) analysis of the freshly prepared test preparations showed amount of carbon present within the control and the 100 mg/l loading rate WAF test vessels to be less than the limit of quantitation (LOQ) for the method within fresh and old media. Therefore the results from the TOC analysis did not provide definitive evidence of the stability of the test preparations.

The test results, expressed as the EL50 value, showed that the sample was not hazardous to Daphnia magna at a loading rate of 100 mg/l.

Conclusion

Short-term Daphnia toxicity tests are available for the registered substance and for two GTL-derived substances in the relevant carbon number range for the registered substance. In the absence of adverse effects in these studies it can be concluded that, based on weight of evidence, the ELR50 (freshwater invertebrates) for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics is >100 mg/l.