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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
5th August 2014 to 13th August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP guideline study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
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:
Procedure
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
Key result
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: The toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole.
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
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 wth 95 % confidence limits of 0.75 – 1.0 mg/L.
The 48h EC50 was 0.71 mg/L wth 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 GS250 (Hydrocarbons, C14-C16, 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/lL; 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 EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
5th August 2014 to 13th August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
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
Key result
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: The toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole.
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
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 EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
4th August 2008 to 7th September 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented guideline study. NOELR according to OSPAR COM (not relevant in the case of acute toxicity test according to EC standards). Based on WAFs. No analytical monitoring.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: ISO 14669 - 1999 Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)
Deviations:
no
Principles of method if other than guideline:
guideline study
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No test surrogate material is tested
Analytical monitoring:
no
Details on sampling:
Not applicable (no analytical monitoring)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Water accomodation fractions (WAF) were prepared individually by stirring appropriate amounts of the test substance in natural seawater according to ISO 14442-2006. The solutions were stirred with a spin bar for 22 hrs at a speed which formed a vortex one-third of the depth of the fluid content, followed by standstill for 1.5 hrs. Closed aspirator bottles were used and samples for testing were drawn off through a drain port near the bottom of the bottle.
For two of the concentrations, 3.0 and 99.2 mg/L WAF the spin bar had stopped during the night. They were turned again for 1 hour in the morning. It is not known for how long the spin bars stood still.

-Seawater: natural seawater from an unpolluted site at about 80 m depth in Byfjord, collected from a tap at Akvamiljo on 11.06.2008, after filtration through a sand filter. This seawater had a salinity of 34.8 per thousand. At the lab, the water was filtered through a GF/C filter and briefly heated to 75°C prior to use. In this test seawater with addition of nutrients (according to ISO 110253 - 2006) was used since an algae test was also conducted.
Test organisms (species):
other aquatic arthropod: Acartia tonsa
Details on test organisms:
TEST ORGANISM
Acartia tonsa, Marine planktonic copepod

- Source: bought as eggs from Marin Biologiisk laboratorium, Kobenhaven University, Helsingor.
- Age at study initiation: 15 days
- Feeding during test: no feeding

ACCLIMATION
The test organisms used in the test were hatched 21.07.2008 at M. Lab and held at a temperature of 19.5 - 24.1 °C. They were fed with a culture of Rhodomonas baltica from day 2.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observation
Hardness:
Not relevant (seawater study)
Test temperature:
19.5 - 21.5 °C
pH:
8.3
Dissolved oxygen:
7.0 - 7.4 mg/L
Salinity:
34.8 per thousand
Nominal and measured concentrations:
Nominal concentrations (loadings): 3.0, 10.0, 31.7, 99.2, 319, 1038, and 3193 mg/L (WAF)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: 28 mL glass rolled rim vials each containig 25 mL of test solution
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): no renewal (static test)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 6


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural seawater from an unpolluted site at about 80 m depth in Byfjord,filtered through a sand filter, then through a GF/C filter and briefly heated to about 75°.


OTHER TEST CONDITIONS
- Photoperiod: 16 hrs light / 8 hrs dark


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : lethality after 24 and 48 hrs


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.20 (mean)
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
24 h
Dose descriptor:
LL50
Effect conc.:
> 3 193 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading)
Basis for effect:
mortality
Remarks on result:
other: WAF
Key result
Duration:
48 h
Dose descriptor:
LL50
Effect conc.:
> 3 193 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading)
Basis for effect:
mortality
Remarks on result:
other: WAF
Duration:
24 h
Dose descriptor:
other: LL90
Effect conc.:
> 3 193 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading)
Basis for effect:
mortality
Remarks on result:
other: WAF
Key result
Duration:
48 h
Dose descriptor:
other: LL90
Effect conc.:
> 3 193 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading)
Basis for effect:
mortality
Remarks on result:
other: WAF
Details on results:
- Mortality of control: 10%
Results with reference substance (positive control):
- Mortality: 66.7%

Nominal loading

(mg/L)

Dissolved oxygen

(mg/L)

pH

Control

7.4

8.3

3.0

7.1

8.3

10.0

7.2

8.3

31.7

7.2

8.3

99.2

7.1

8.3

319

7.1

8.3

 1038  7.1  8.3
 3193  7.1  8.3

Nominal Loading

(mg/L)

Total number immobilized

(24 hrs)

(48 hrs)

Control

1

3

3.0

0

3

10.0

0

2

31.7

1

6

99.2

0

7

319

0

2

 1038  0  4
 3193  0  3

Validity of the test: the acceptance criteria were fulfilled

- All copepods were from the same population

- The percentage mortality in the blank did not exceed 10%

- The 48-hr mortality obtained after exposure to 1.0 mg/L 3,5 -dichlorophenol was 66.7 % (in the expected range of 20%-80%)

- The oxygen concentration was greater than 4 mg/L throughout the test (7.0 - 7.4 mg/L)

Validity criteria fulfilled:
yes
Remarks:
Copepods all from the same population ; mortality in blanks <=10%; oxygen concentration >4mg/L; 48-hr mortality with reference substance within 20-80%
Conclusions:
48-hr LL50 > 3193 mg/L (based upon nominal loading rate)
Executive summary:

An acute toxicity test was conducted on the marine copepod Acartia tonsa. The test substance concentrations (nominal loading rates) ranged from 32.9 to 3190 mg/L. Test solutions were prepared as individual water accomodation fractions (WAF). Control and dilution water were natural seawater from an unpolluted site at about 80m depth in Byfjord, filtered through sand filter at the collection, then through a GF/C filter and briefly heated to 75°C prior to use. Test substance was mixed in dilution water for 22 hours, and the mixture was allowed to settle for 1.5 hours prior to drawing off the aqueous solutions for testing. Test vessels were glass 28 mL rolled rim vials, each containing 25 mL of test solution. In the test, 5 concentrations were tested in a geometric series: 32.9, 99.3, 320, 1000, and 3190 mg/L. Four replicates for the test substance and six for the blank were used, with 5 copepods per vial.

Dissolved oxygen was >4 mg/L during the study. The pH was 8.1 - 8.2. The temperature was 20.3 - 21.9 °C.

Test organisms were obtained from eggs. At test start, the test organisms were 15 days (after hatching).

No excursions from protocol were noted.

Analytical monitoring was not performed.

48-hr LL50 > 3193 mg/L (based upon nominal rate)

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
March to November 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
An equilabration study was conducted prior to the study to determine the length of time needed to develop water accommodated fractions (WAFs) at the 1000 mg/L loading level. The aqueous phase of the WAF was analyzed after 1, 24, 48, and 96 hours of stirring. In all samples, the quantity of test substance in solution was less than the limit of detection (0.5 mg/L). Further analysis of test substance in test media a loading level of 1 g/L also showed no detectable amounts of test substance. Consequently, analysis of treatment solutions from the definitive study were not conducted.
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
Organisms used in the test were from a laboratory culture were less than 24 hour old. The source of the culture was from ICI Brixham Laboratory from I.R.Ch.A., France.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
206 mg/L CaCO3
Test temperature:
18 to 19 degrees C
pH:
6.9 to 7.4
Dissolved oxygen:
9.0 to 9.1 mg/L
Salinity:
Reshwater medium
Nominal and measured concentrations:
The nominal loading rates were 10, 30, 100, 300, and 1000 mg/L. A control was also tested.
Details on test conditions:
The study was conducted in closed test systems that were not renewed during the study. The test systems used were 150 ml glass Erlenmeyer flasks. The control and treatment levels were evaluated in replicate test systems. Ten organisms, less than 24 hours old, were exposed in each of the control and treatment solution flasks and were not fed during the study.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
LL0
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LL0
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
There was no immobility reported in any of the treatment solutions and control at test termination.
Reported statistics and error estimates:
None
Validity criteria fulfilled:
yes
Conclusions:
The water accommodated fraction (WAF) of the test substance did not produce a 50% effect (immobility) with Daphnia magna at a loading of 1000 mg/L after a 48-hour exposure. Therefore, the 48-hour LL50 is reported as >1000 mg/L. There was no mortality at the 1000 mg/L loading level after 48 hours. Therefore, the 48-hour LL0 for mortality is reported as 1000 mg/L. There was also no mortality in the control.
Executive summary:

The water accommodated fraction (WAF) of the test substance did not produce a 50% effect (immobility) with Daphnia magna at a loading of 1000 mg/L after a 48-hour exposure. Therefore, the 48-hour LL50 is reported as >1000 mg/L. There was no mortality at the 1000 mg/L loading level after 48 hours. Therefore, the 48-hour LL0 for mortality is reported as 1000 mg/L. There was also no mortality in the control.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
October 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 2 because only limited analyses accompanied the study. However, the study generally followed a standard guideline and GLP, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 850.1020 (Gammarid Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The water accommodated fraction prepared at 10000 mg/L and the control used on Day 3 of the study were analyzed. Both samples were analyzed upon preparation and after 24 hours.
Vehicle:
no
Details on test solutions:
Individual treatment solutions were prepared by adding accurately weighed test substance, to 1.0 L of natural seawater in erlenmeyer flasks. The stock solutions were thoroughly mixed and the phases were allowed to separate for 24 hours. The Water Accommodated Fraction (WAF) was then drawn off the mixing vessel into the test vessels.
Test organisms (species):
other: Chaetogammarus marinus
Details on test organisms:
Test organisms were cultured in the laboratory prior to use in the test. They had a mean length of 4.2 mm. During the test, each test organism was fed a few Artemia nauplii per day.
Test type:
semi-static
Water media type:
saltwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
15.0 degrees C (mean)
pH:
7.8 to 7.9 for new solutions and 7.7 to 8.0 for 24-hour old solutions.
Dissolved oxygen:
7.7 to 9.1 mg/L for new solutions and 6.8 to 8.5 mg/L for 24-hour old solutions.
Salinity:
33.7‰ (3.4%)
Nominal and measured concentrations:
Nominal treatment levels included the control and 10,000 mg/L. Measured concentrations were determined only for the treatment level on day 3 for the freshly prepared treatment solution and after 24 hours upon renewal. The 10,000 mg/L treatment solution analytical results were only reported for the normal paraffinic constituents, which totaled greater than the limit of detection (0.004 mg/L) in the freshly prepared exposure solution and at the limit of detection, 0.004 mg/L, in the 24-hour old exposure solution. The purpose of the analyses was to demonstrate that components of the test substance were in solution, because no effects were demonstrated in the study.
Details on test conditions:
Test vessels were closed scintillation vials filled with approximately 20 mL of test solution. Twenty replicates each of the treatment and control were tested, each containing 1 test organism. The test organisms were gently transferred daily to vials with freshly prepared WAF. Lighting was 16 hrs light and 8 hrs dark.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Swinning behavior and food uptake
Details on results:
Mortality results for the definitive test:
Nominal Crustacean Total
Loading Mortality Mortality
(mg/L) (@ 24, 48, 72, 96 hrs)* (%)
Control 0, 0, 0, 0 0
10,000 0, 0, 0, 0 0

Mortality results for the range-finding test:
Nominal Crustacean Total
Loading Mortality Mortality
(mg/L) (@ 24, 48, 72, 96 hrs)* (%)
Control 0, 0, 0, 0 0
1 0, 0, 0, 0 0
100 0, 0, 0, 0 0
1000 0, 0, 0, 0 0

*20 organisms tested at each of the control and treatment levels
Reported statistics and error estimates:
Statistical analyses were not conducted.
Validity criteria fulfilled:
yes
Conclusions:
The water accommodated fraction (WAF) of the test substance did not produce a 50% effect (mortality) with Chaetogammarus marinus at a loading of 10,000 mg/L after a 96-hour exposure. Therefore, the 96-hour LL50 is reported as >10,000 mg/L. There was no mortality at the 10,000 mg/L loading level after 96 hours. Therefore, the 96-hour NOELR for mortality is reported as 10,000 mg/L. There was also no mortality in the control.
Executive summary:

The water accommodated fraction (WAF) of the test substance did not produce a 50% effect (mortality) with Chaetogammarus marinus after 96 hours at a loading of 10,000 mg/L, the only treatment level tested. Therefore, the 96-hour LL50 is reported as >10,000 mg/L. There was no mortality at the 10,000 mg/L loading level after 96 hours and no effects on swimming and feeding behavior were observed. Therefore, the 96-hour NOELR for these endpoints is reported as 10,000 mg/L. There was also no mortality in the control during the study. The control and treatment solutions were renewed every 24 hours. Analytical results showed that the treatment solution contained components of the test substance and that their combined concentration was greater than the limit of detection of the test substance, based on one sample from a freshly prepared WAF for day 3. The only components of the test substance quantitated were the normal paraffins. Threrefore, the analytical data do not characterize the solubility of the test substance from the 10,000 mg/L loading, but only serve to demonstrate that components of the test substance were in solution. The concentration of the test substance from the day three 24-hour old sample was reported at the limit of detection. The low analytical results are to be expected because of the very low water solubility of the components of the test substance. The day 3 analyses were conducted because no effects were observed during the first two days of the study and confirmation of exposure to the test substance was needed.

Description of key information

There is no data available for this substance. However, key and supporting data is available for structural analogues. The data is read across to this substance based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13. Key information is summarised below:

Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics presented a 48-h EL50 for Daphnia magna of >100 mg/L (WAF) based on mobility.

Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics presented a 48-h EL50 for Daphnia magna of >100 mg/L (WAF) based on mobility.

Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 48-h LL50 for Acartia tonsa of > 3193 mg/L (based upon nominal loading rate).

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 48-h LL50 for Daphnia magna of >1000 mg/L.

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics presented a 96-h LL50 for Chaetogammarus marinus of >10,000 mg/L.  

No LC50 or LL50 could be derived from the acute toxicity studies for aquatic invertebrates. The toxicity level was never reached in any study despite the high loading level tested. Therefore, this substance is considered to display no acute toxicity to aquatic invertebrates based on an analogue read across approach.

Key value for chemical safety assessment

Additional information

The substances are 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 (EL50 or LL50). No EC50 or LC50 could be derived from the acute toxicity studies.

The water accommodated fraction (WAF) of Hydrocarbons, C14-C16, n-alkanes, isoalkanes, <2% aromatics did not produce a 50% effect (mobility) with Daphnia magna at a loading of 100 mg/L after a 48-hour exposure. Therefore, the 48-hour EL50 is reported as >100 mg/L and the 48-hour NOELR for mobility is reported as 100 mg/L.

The water accommodated fraction (WAF) of Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics did not produce a 50% effect (mobility) with Daphnia magna at a loading of 100 mg/L after a 48-hour exposure. Therefore, the 48-hour EL50 is reported as >100 mg/L and the 48-hour NOELR for mobility is reported as 100 mg/L.

The water accommodated fraction (WAF) of Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics did not produce a 50% effect (mortality) with Chaetogammarus marinus at a loading of 10,000 mg/L after a 96-hour exposure. Therefore, the 96-hour LL50is reported as >10,000 mg/L. There was no mortality at the 10,000 mg/L loading level after 96 hours. Therefore, the 96-hour NOELR for mortality is reported as 10,000 mg/L. There was also no mortality in the control.

The water accommodated fraction (WAF) of Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics did not produce a 50% effect (immobility) with Daphnia magna at a loading of 1000 mg/L after a 48-hour exposure. Therefore, the 48-hour LL50is reported as >1000 mg/L. There was no mortality at the 1000 mg/L loading level after 48 hours. Therefore, the 48-hour LL0 for mortality is reported as 1000 mg/L. There was also no mortality in the control.

Many study reports that investigated the toxicity of the substance Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, ≤2% aromatics on a marine copepod, Acartia tonsa exposed to water accommodated fractions (WAFs) are available. Several studies indicate very high values (up to 69155 mg in Altin, 1998b), In most of the studies the calculation of LL50could not be achieved because of the low mortality levels obtained (Aunaas, 1996a and c; Larsen, 2006a, b, d and e; 2008a-c). When more than 5 values are available for LL50, the geometric mean of all the values is considered for LL50. However, in this particular case, as a great number of studies could not achieve the calculation of LL50, the geometric mean cannot be calculated, but 9 studies among 11 found that LL50was greater than 3000 mg/L. Therefore, it is considered that LL50is >3000 mg/L.