Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 January 1995 and 31 March 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity for poorly water soluble complex mixtures, the Sponsor requested modification of the standard methods for the preparation of aqueous media. Methods involving the continuous stirring of complex mixtures, like oil products have been used, particularly for exposing robust organisms like fish. Such approaches are unsuitable for small organisms like Daphnia and algae, which will be damaged by the turbulence and physically fouled by undissolved material recent evidence has suggested that for some products, physical fouling of fish may also lead to inconsistent results. As an alternative, the use of solvents or surfactants to solubilise or disperse poorly soluble products has been widely advocated in the past. This approach results in the production of aqueous media in which the test material is in a different form from that encountered following accidental release unto the environment. In addition, there may be interaction between the surfactant/solvent and the product. The approach recommended by CONCAWE for oil products (1) and subsequently endorsed by several important regulatory authorities in the EC (2,3) and elsewhere, is to expose organisms to the “Water Accommodated Fraction” (WAF) of the oil product. Using this approach, aqueous medium is prepared by mixing the product with water for a prolonged period (usually 24 hours), with continuous stirring to ensure equilibration between the product and water phases. At the completion of mixing, the product phase is separated and organisms exposed to the aqueous phase (which will contain dissolved material, along with any part of the product that may be present in emulsified or dispersed form). Exposures are expressed in terms of the original concentration of product in water at the preparation of WAF (the loading rate), irrespective of the actual concentration of product dissolved in the water.
GLP compliance:
yes (incl. certificate)
Remarks:
The work described was performed in compliance with the UK Principles of Good Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989).

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Standard solutions were prepared in hexane at nominal concentrations of 10, 100, 1000 and 10,000 mg/l.

- Sampling method:
An aliquot (500ml) of each test sample was extracted with three portions (3 x 50 ml) of dichloromethane, each extract being filtered through a bed of anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue dissolved in hexane (2.0ml)


- Sample storage conditions before analysis:
Not applicable, samples used immediately.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
For the purpose of the definitive study the test material was prepared as a Water Accommodated Fraction.

Approximately 24 hours prior to the study start an amount of test material (2.0 g) was dispensed onto the surface of 2 litres of reconstructed water to give a 1000 mg/l loading rate and stirred for 20 hours. The stirrer rate (rpm) of the magnetic stirrer and the depth of the vortex (approximately 20-25% of depth of the mixing vessel) were recorded after 20 hours stirring was stopped and the mixture allowed to stand for 4 hours prior to removal of the aqueous phase or Water Accommodated Fraction was dispersed into four replicate test vessels, each containing 200 ml of test culture.

- Eluate:
Not applicable

- Differential loading:
Not recorded

- Controls:
Control vessel contained 200mL of the test media without being exposed to the test material

- Chemical name of vehicle:
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material:

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name:
Daphnia

- Strain:
Not applicable

- Source:
Maintained in laboratory culture originating from a strain supplied by Institut National de Recherche Chimique Appliquee (I.R.CH.A).

- Age at study initiation:
1st Instar

- Weight at study initiation:
Not applicable

- Length at study initiation:
Not applicable

- Valve height at study initiation, for shell deposition study:
Not applicable

- Peripheral shell growth removed prior to test initiation:
Not applicable

- Method of breeding:
Culture conditions ensured that reproduction was by parthenogensis. Gravid adults were isolated 24 hours prior to the initiation of the test, the young daphnids produced overnight were then removed for testing.

- Feeding during test
- Food type: No food during exposure
- Amount: No food during exposure
- Frequency: No food during exposure


ACCLIMATION
- Acclimation period:
Not recorded

- Acclimation conditions (same as test or not):
Not recorded

- Type and amount of food:
Each culture was fed daily with a suspension of mixed algae (predominantly Chlorella spp).

- Feeding frequency:
Daily

- Health during acclimation (any mortality observed):
No mortality recorded


QUARANTINE (wild caught)
- Duration:
Not applicable

- Health/mortality:
Not applicable

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
h
Post exposure observation period:
Not recorded.

Test conditions

Hardness:
The reconstituted water has an approximate theoretical total hardness of 270 mg/L as CaCO3.
Test temperature:
Water temperature was recorded at 0, 24 and 48 hours using a thermometer and in a test vessel every 1.6 minutes using a Tinytalk temperature logger.
The temperature was maintained at 20 +/- 1 deg C throughout the study (please see attachment 1)
pH:
pH was recorded at the start and termination of the study.

There were no treatment related differences for pH.
Dissolved oxygen:
Dissolved oxygen was recorded at the start and termination of the study.

There were no treatment related differences for oxygen concentration.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal concentration
Range-finding study: 100 and 1000 mg/l
Definitive study: 100 mg/l

Quantitation of the test and control samples by GC-MS shows that the solubility of the many components of the test material in aqeous media is lower than the limit of quantifitation. Therefore the test concentrations for the Acute Toxicity to Daphnia could not be verified by chemical analysis.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed - covered to reduce evaporation.
- Material, size, headspace, fill volume: 250ml glass jar containing approximately 200 ml of Water Accommodated Fraction
- Aeration: The test vessels were not aerated

- Type of flow-through:
Not applicable

- Renewal rate of test solution:
The Water Accommodated Fractions were not renewed during the exposure period.

- No. of organisms per vessel:
At the start of the study 10 daphnids were placed in each test and control vessel at random

- No. of vessels per concentration (replicates):
4

- No. of vessels per control (replicates):
2

- No. of vessels per vehicle control (replicates):
Not applicable

- Biomass loading rate:
Not recorded.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted water

i) Stock solutions
a) CaCl2.2H2O : 11.76 g/L
b) MgSO4.7H2O : 4.93 g/L
c) NaHCO3 : 2.59 g/L
d) KCl : 0.23 g/L

ii) Preparation
25ml of each of solutions 1 - d were added to each litre (final volume) of deionised water.
The reconstructed water was aerated until the dissolved oxygen concentration was approximately air-saturation value.

- Total organic carbon:
The concentration of Total Organic Carbon (TOC) in the test samples from the Water Accommodated Fraction (WAF) of the test material was determined by Total Organic Carbon analysis.

Approximately 20 ml of the 1000 mg/L loading rate Water Accommodated Fraction was taken for analysis at 0 and 48 hours.

Standard solutions of potassium hydrogen phthalate were prepared in deionised reverse osmosis water at a nominal concentration of 50 mg carbon/litre and used for calibration of the carbon analyser.

The standard and sample solutions were analysed for TOC using a Dohrmann DC-190 high temperature Total Organic Carbon Analyser or an Ionics 1555D high temperature Total Organic Analyser using the following conditions:

a) Dohrmann DC-190
Total Organic Carbon Channel
Temperature : 900 deg C
Carrier gas : Zero grade oxygen
Carrier gas flow rate : 200 mL/min regulated at 50 psi
Catalyst : Platinum
Injection volume : 250 microL

Inorganic Carbon Channel
Temperature : 20 deg C
Carrier gas : Zero grade oxygen
Carrier gas flow rate : 200 mL/min regulated at 50 psi
Catalyst : 20% phosphoric acid
Injection volume : 250 microL

b) Ionics 1555B
Total Carbon Channel
Temperature : 900 deg C
Carrier gas : High purity nitrogen
Carrier gas flow rate : 50 mL/min regulated at 16 psi
Catalyst : Copper oxide
Injection volume : 100 micoL

Inorganic Carbon Channel
Temperature : 150 deg C
Carrier gas : High purity nitrogen
Carrier gas flow rate : 50 mL/min regulated at 16 psi
Catalyst : orthophosphoric acid
Injection volume : 40 micoL

The results of the Total Organic Carbon analysis generated for the test samples taken throughout the study have been detailed in Table 2.

- Particulate matter:
None recorded

- Metals:
K and Mg present in the reconstitued water

- Pesticides:
None recorded

- Chlorine:
KCl 0.23 g/L

- Alkalinity:
pH equal to 7.8 +/- 0.2

- Ca/mg ratio:
CaCl2.2H2O 11.76 g/L : MgSO4.7H2O : 4.93 g/L

- Conductivity:
Conductivity <5 microS cm-1

- Culture medium different from test medium:
No

- Intervals of water quality measurement:
Not recorded

OTHER TEST CONDITIONS
- Adjustment of pH:
pH adjusted (if necessary) with NaOH or HCl.

- Photoperiod:
16 hours light, 8 hours darkness for a period of 48 hours

- Light intensity:
Not recorded

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Water samples were taken from the control and 1000 mg/L loading rate Water Accommodated Fraction test group (replicates R1 plus R2 and R3 plus R4 pooled) at 0 and 48 hours for Total Organic Carbon (TOC) analysis.

Compound specific analysis was not performed on the test preparation due to the concentration of the test material in the Water Accommodated Fractions being below the limit of detection of the analytical method.

An estimate of the ELR 50 values was given by inspection of the immobilisation data.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Range finding study : x 10
Definitive study : not applicable.

- Justification for using less concentrations than requested by guideline:
- Range finding study
The test concentrations to be used in the definitive study were determined by a preliminary range-finding study.

In the range-finding study Daphnia magna were exposed to a series of 100 and 1000 mg/L Water Accommodated Fractions of the test material at loading rates of 100 and 1000 mg/L.

For the purpose of the range-finding study, amounts of test material (0.20 and 2.00 g) were each separately dispersed onto the surface of 2 litres of reconstituted water to give 100 and 1000 mg/L loading rates respectively and then stirred by magnetic stirrer for 24 hours prior to the study start, care was taken to avoid vortex formation or gross mixing. Stirring was stopped after 24 hours and the mixture allowed to stand for 1 hour prior to removal of the aqueous phase or Water Accommadated Fraction (WAF for testing). The Water Accommodated Fractions were not prepared by stirring the test water to give a vortex of 20 - 25% of the water column height due to the range-finding study being carried out prior to the request of the Sponsor to carry out mixing with the production of a vortex and a mixing period of 20 hours and 4 hours standing period.

- Test concentrations:
100 and 1000 mg/L

- Results used to determine the conditions for the definitive study:
Based on the results of the range-finding study a "Limit test" was conducted for the definitive study at 1000 mg/L loading rate Water Accommodated Fraction to confirm that at the maximum test concentration defined by the Sponsor no immobilisation or adverse reactions to exposure were observed.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
other: ELR50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: loading rate Water Accommodated Fraction
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: loading rate Water Accommodated Fraction
Details on results:
- Behavioural abnormalities:
There were no adverse reactions to exposure.

- Observations on body length and weight:
Not applicable

- Other biological observations:
Immobilisation data
There was no immobilisation in 40 daphnids exposed to a 1000 mg/L loading rate Water Accommodated Fraction for a period of 48 hours. Inspection of the immobilisation data gave the following results:

Time (h) ELR*50 (mg/L) 95% Confidence Limits (mg/L)
24 >1000 -
48 >1000 -

*ELR = Effective Loading Rate. These results are based on effective loading rate of Water Accommodated Fractions

The No Observed Effect Concentration after 24 and 48 hours exposure were greater than or equal to 1000 mg/L loading rate Water Accommodated Fraction. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L loading rate Water Accommodated Fraction.
- Mortality of control:
No mortality observed

- Other adverse effects control:
Not applicable.

- Abnormal responses:
There were no adverse reactions to exposure

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
The test concentrations for the Daphnia study could not be verified by GC analysis.

- Effect concentrations exceeding solubility of substance in test medium:
Observations on test material solubility were carried out during the mixing and testing of the Water Accommodated Fraction.

At 24 hours prior to the study start, at the start of the mixing period, the test material was observed to be contained within the vortex and present as clear, oily globules on the water surface. However, after 20 hours of stirring and 4 hours of standing the test material was observed at the water surface only. During testing, the Water Accommodated Fraction was observed to be a clear, colourless solution at 24 and 48 hours.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
None recorded

Any other information on results incl. tables

The stirrer rate (rpm) of the magnetic stirrer and the vortex depth were recorded at the start and end of the mixing period (See Table 1 below).

Table 1: Stirrer Rate and Vortex Depth at the Start and End of the Mixing Period

  Nominal Loading Rate (mg/L)
Control 1000
 - 24 hours  - 4 hours  - 24 hours  - 4 hours
Height of Water Column (cm) 15 15 15 15
Depth of Vortex (cm) 3.5 3.4 3 3.1
% of Depth of Mixsing Vessel 23.3 22.7 20 20.7
Stirrer rate (rpm) 600 600 660 650

Table 2: Results From Total Organic Carbon (TOC) Analysis

Samples Nominal Loading Rate (mg/L) Concentratio of TOC (mg C/L) Concentration of TOC corrected for control (mg C/L)
0 hours Control 0  - 
1000 R1 - R2 0.67 0.67
1000 R3 - R4 0 0
48 hours Control 13.48  - 
1000 R1 - R2 1.04 < Control
1000 R3 - R4 0.9 < Control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity to Alkane 2 to the freshwater invertebrate Daphnia magna has been investigated and gave a 48 hour ELR50 of greater than 1000 mg/L loading rate Water Accommodated Fraction. Correspondingly the No Observed Effect Concentration was greater than or equal to 1000 mg/L loading rate Water Accommodated Fraction.
Executive summary:

1. Methods

A study was performed to assess the acute toxicity of the test material, Alkane 2, to Daphnia magna . The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No. 202, "Daphnia sp., Acute Immobilisation Test and Reproduction Test" referenced as Method C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and US EPA Code of Federal Regulations, Title 40, Part 797, Section 1300.

2. Procedures

Following a preliminary range-finding study, forty daphnids (4 replicates of 10 animales) were exposed to a Water Accommodated Fraction of the test material at a loading rate of 1000 mg/L for 48 hours under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

3. Results

The 48 hour ELR*50 for the test material to Daphnia magna based on nominal loading rates was greater than 1000 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 1000 mg/L.

*ELR50 = Effective Loading Rate.