[No public or meaningful name is available]

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 23 March 2009 and 17 April 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Previous experience gained from studies conducted on poorly water soluble test materials has shown that a mixing period of 24 - 48 hours is sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/l to confirm that no immobilisation or
adverse reactions to exposure were observed.

Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control (replicates R1 - R4 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 - R2 and R3 - R4 pooled) at 0 and 48 hours (see Appendix 1 in attached section).

Duplicate samples were taken and stored at approximately -20 degree C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances in the WAF. A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis (see Appendix 2 in attached section).

Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).

An amount of test material (1100 mg) was added to the surface of 11 litres of reconstituted water to give the 100 mg/l loading rate. After the
addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a
slight dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube,
covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the
vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal and the WAF removed by mid-depth
siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic observations of the WAF were
performed and showed there to be no micro-dispersions of test material present.

The test material is a complex mixture and hence concentrations in the test preparations were not determined by analysis at the request of the
Sponsor.

Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period and after a 1-Hour standing period the WAF was observed to have formed a clear colourless water column with test material floating on the surface. Microscopic examination of the WAF showed there to be no micro-dispersions of test material present. After siphoning and for the duration of the test, the 100 mg/l loading rate was observed to be a clear, colourless solution.

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 polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 degree 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 suspension of algae (Chlorella sp.). 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.


The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

The reconstituted water is defined below:

Reconstituted Water
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
i
Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was
approximately air-saturation value.

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test type:

static

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not Applicable

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 21ºC throughout the test - see table of physico measurements in section in any other information for measurements.

pH:

The pH was measured at the start and termination of the test - see table of physico measurements in section in any other information for
measurements.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test - see table of physico measurements in section in any other
information for measurements

Salinity:

Not Applicable

Nominal and measured concentrations:

Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/l to confirm that no immobilisation or
adverse reactions to exposure were observed.

Details on test conditions:

In the definitive test 250 ml glass jars containing approximately 200 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 covered to reduce evaporation and maintained in a temperature controlled room at 21ºC with a photoperiod of 16 hours light 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 material.

The test preparations were not renewed during the exposure period. Any immobilisation 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 immobilised 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: Not stated

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: Not stated

Details on results:

RESULTS
Validation of Mixing Period
Pre-study work (see Appendix 2 in attached section) indicated that there was no significant increase in the amount of total organic carbon by
extending the preparation period for longer than 24 hours.

Range-finding Test
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1 -see
section any other information on results .

No immobilisation was observed at the 1.0, 10 and 100 mg/l loading rate WAFs.

Based on this information, a single loading rate of four replicates, of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.

Definitive Test
Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2 see sectionany other information on results.

There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours. Inspection of the immobilisation
data gave the following results:
Time (h) EL*50 (mg/l) 95% Confidence limits
(mg/l)
24 >100 -
48 >100 -

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is
based upon zero immobilisation at this loading rate.

Evaluation of data
An estimate of the EL*50 values was given by inspection of the immobilisation data.

Results with reference substance (positive control):

Positive Control

The positive control (Harlan Laboratories Ltd Project Number: 0039/1069) was conducted between 09 December 2008 and 11 December 2008.

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No: 0039/1069) during the positive control are given in Table 3 - see section any other information on results.

The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2 - see attachement Appendix-3)

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:

Time (h)
EC50 (mg/l) 95% Confidence limits
(mg/l)
3 > 3.2 -
24 0.82 0.71 - 0.94
48 0.71 0.61 - 0.81

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero
immobilisation at this concentration.

The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (SE = 1.7) and 8.6 (SE = 1.8) respectively.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.21).

Table1: Cumulative Immobilisation Data in the Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	0
10	0	0
100	0	0

Table 2: Cumulative Immobilisation Data in the DefinitiveTest

Nominal Loading Rate (mg/l)		Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)
		24 Hours			48 Hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
Control	R2	0			0
Control	R3	0			0
Control	R4	0			0
100	R1	0	0	0	0	0	0
100	R2	0			0
100	R3	0			0
100	R4	0			0

R₁– R₄= Replicates 1 to 4

Table 3: Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	1	1	2	10	2	2	4	20
1.0	0	0	0	0	7	8	15	75	9	9	18	90
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	0	0	0	0	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ to

Daphnia magna. The method followed that described in 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.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 21ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EL^[1]₅₀for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l. The test material is a complex mixture and hence compound specific analysis of the WAFs was not performed. However samples of the control and 100 mg/l loading rate WAF were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis. Given the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved organic carbon concentrations were below the limit of quantitation at 0 and 48 hours, the results were based on nominal loading rates only.

The 48-Hour EC₅₀for the reference material to Daphnia magna based on nominal concentrations was 0.71 mg/l with 95% confidence limits of 0.61 – 0.81 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

[1] EL = Effective Loading rate

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26, without GLP and analytics but acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not specified

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Water accommodated fractions (WAF’s) were prepared in sealed vessels with a small headspace according to the CONCAWE methodology for the ecotoxicological testing of petroleum products (CONCAWE report, 1993). WAF’s were prepared using 1L reconstituted water (ISO 6341-1982). The required amount of test substance was weighed on microscope glass cover slips and added to 1L media. The vessels were sealed and vigorously shaken (200 rpm’s) for 24 hours on an orbital shaker. The mixtures were poured into separation funnels and left for two hours to separate. The water fractions were transferred to other vessels and used in the test.

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test temperature:

22±2°C

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: water accommodated fraction

Table 1:

Acute toxicity of Diesel fuel to Daphnia magna 

 

Diesel Fuel	NOEL (mg/L)
Test material	>1000
FCC simulated crude derived refinery	>100
Test material+ FCC	>100
DHC simulated crude derived refinery	>1000
Test material+ DHC	>100

The test material and the DHC refinery simulated crude did not adversely affect the test organisms at 1 000 mg/L, indicating that the NOEL were >1 000 mg/L. The FCC refinery simulated crude, DHC+ test material and FCC+ test material blends did however, adversely affect theD. magnaat 1 000 mg/L, but less than 50% of the test organisms were immobilised, indicating that the NOEL of the diesel fuels are > 100 mg/L.

Validity criteria fulfilled:

not specified

Conclusions:

The test substance did not adversely affect the test organisms at 1000 mg/L, indicating that the NOEL were > 1000 mg/L (nominal).

Executive summary:

The test was conducted in accordance with the OECD Guidelines No. 202. The swimming capability or immobilisation of D. magna was used as the endpoint in determining the acute toxicity to the diesel products. D. magnaneonates (<24 hours old) were exposed to the WAF’s (100mg/L and 1000mg/L) of the diesel products over a period of 48 hours. Exposures were performed in quadruplicate using five animals in each replicate. Immobilised D. magna were recorded at 24 and 48 hours of the test. 

The test substance did not adversely affect the test organisms at 1000 mg/L, indicating that the NOEL were >1000 mg/L (nominal).

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26; no information on GLP, but the test parameters documented are sufficient to accept the data

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable, since neither a surrogate nor an analogue material was used in the test.

Analytical monitoring:

no

Details on sampling:

- Concentrations:
Loading rates (mg/L):
I. 100% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear': 1000,
II. 76% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 24% ULSD: 100, 220, 460, 1000.
II. 76% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 24% ULSD: 100, 220, 460, 1000.
III. 50% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 50% ULSD: 100, 220, 460, 1000.
IV. 24% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 76% ULSD: 100, 220, 460, 1000.
V. 100% ULSD: 1, 3, 10, 30, 100, 300, 1000.
VI. Anullex BX: 1000.
- Sampling method: After 24 and 48 hours the numbers of immobilised daphnids were sampled.
- Sample storage conditions before analysis: Not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test samples are mixtures containing a number of moderately volatile components with a range of relatively low water solubilities.
Therefore water accommodated fraction (WAF) methodology was used for production of test media. Vessels containing the medium and test sample
were sealed, leaving only a small headspace, and the contents stirred at 150 rpm for approximately 72 hours (+/- 2 hours). After stirring, the
contents of the vessels were left to settle (1-2 hours) to allow the undissolved material to separate out.
- Eluate: Not applicable (static test)
- Differential loading: Not applicable
- Controls: Control media were subject to the same regime but did not contain the test sample.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not observed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, taken from a clonal laboratory culture
- Age at study initiation (mean and range, SD): Less than 24 hours old

ACCLIMATION
- Acclimation period: No information available
- Acclimation conditions (same as test or not): For the first 14 days the D. magna are cultured in 1 L Pyrex glass beakers containing 0.8 L of
reconstituted freshwater (Appendix B). From Day 15 onwards they are cultured in a 2 litre beaker containing 1.5 litre of reconstituted freshwater. The cultures are held in a temperature controlled room, nominally 18-22°C, under artificial light in a 16 h light 8 h dark cycle.
- Type and amount of food: The cultures are fed daily with a concentrated suspension of Chlorella vulgaris to give a concentration of approximately
0.10 x 106 cells/mL. The C. vulgaris is obtained from a 4 litre culture grown under semi-axenic conditions. Prior to use the C. vulgaris is concentrated by filtration and resuspension in sterilised C. vulgaris growth medium. The C. vulgaris is stored in a refrigerator at 4°C and used within one week.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): No mortality observed

QUARANTINE (wild caught)
- Duration: Not applicable
- Health/mortality: Not applicable

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

Test with 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear':
Total hardness (mg as CaCO3): 174 at test initiation (T = 0 h)
II. Test with ULSD:
Total hardness (mg as CaCO3): 175 at test initiation (T = 0 h),
III. Test with Annulex BX:
Total hardness (mg as CaCO3): 180 at test initiation (T = 0 h),
IV. Test with 50% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 50% ULSD:
Total hardness (mg as CaCO3): 164 at test initiation (T = 0 h),
V. Test with 76% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 24% ULSD:
Total hardness (mg as CaCO3): 174 at test initiation (T = 0 h)

Test temperature:

Test with 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear':
20.0-20.1 °C (reported at hourly intervals)
II. Test with ULSD:
20.0-20.2 °C (reported at hourly intervals),
III. Test with Annulex BX:
20.0-20.2 °C (reported at hourly intervals),
IV. Test with 50% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 50% ULSD:
19.9-20.1 °C (reported at hourly intervals),
V. Test with 76% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 24% ULSD:
20.0-20.1 °C (reported at hourly intervals)

pH:

I. Test with 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear':
pH at test initiation (T = 0 h): 8.2-8.3,
pH at test finalisation (T = 48 h): 8.0-8.1
II. Test with ULSD:
pH at test initiation (T = 0 h): 8.1-8.3,
pH at test finalisation (T = 48 h): 7.6-8.5
III. Test with Annulex BX:
pH at test initiation (T = 0 h): 8.6,
pH at test finalisation (T = 48 h): 7.9-8.6
IV. Test with 50% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 50% ULSD:
pH at test initiation (T = 0 h): 8.6,
pH at test finalisation (T = 48 h): 8.2-8.6
V. Test with 76% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 24% ULSD:
pH at test initiation (T = 0 h): 8.4-8.5,
pH at test finalisation (T = 48 h): 8.1-8.4

Dissolved oxygen:

I. Test with 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear':
Concentration of dissolved oxygen (mg/L):
- at test initiation (T = 0 h): 8.2-8.3,
- at test finalisation (T = 48 h): 7.9-8.0
II. Test with ULSD:
Concentration of dissolved oxygen (mg/l):
- at test initiation (T = 0 h): 8.1-8.3,
- at test finalisation (T = 48 h): 6.9-7.8
III. Test with Annulex BX:
Concentration of dissolved oxygen (mg/L):
- at test initiation (T = 0 h): 8.1-8.2,
- at test finalisation (T = 48 h): 7.0-7.7
IV. Test with 50% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 50% ULSD:
Concentration of dissolved oxygen (mg/L):
- at test initiation (T = 0 h): 8.0-8.3,
- at test finalisation (T = 48 h): 8.6
V. Test with 76% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 24% ULSD:
Concentration of dissolved oxygen (mg/L):
- at test initiation (T = 0 h): 8.2-8.3,
- at test finalisation (T = 48 h): 7.3-7.5

Salinity:

Not relevant

Nominal and measured concentrations:

- Concentrations:
Loading rates (nominal, mg/L):
I. 100% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear': 1000,
II. 76% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear', 24% ULSD: 100, 220, 460, 1000.
II. 76% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear', 24% ULSD: 100, 220, 460, 1000.
III. 50% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear', 50% ULSD: 100, 220, 460, 1000.
IV. 24% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear', 76% ULSD: 100, 220, 460, 1000.
V. 100% ULSD: 1, 3, 10, 30, 100, 300, 1000.
VI. Anullex BX: 1000.

The nominal concentrations were not confirmed by analytical measurements.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (sealed with a screw cap)
- Material, size, headspace, fill volume: 150 mL Erlenmayer flasks
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no renewal of the test media (static test)
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: 10 daphnids
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The water used for the testing is a reconstituted fresh water prepared by dissolving the following amounts of Analar grade salts in reverse osmosis filtered water (recommended for producing "hard" water by US EPA, 1975):
192 mg/L NaHCO3, 120 mg/L CaSO4xH2O, 240 mg/L MgSO4x7H2O, 8 mg/L KCl.
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: information not available

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark
- Light intensity: Artificial light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilisation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: no data
- Results used to determine the conditions for the definitive study: no data

Reference substance (positive control):

yes

Remarks:

AGO - Ultra Low Sulphur Diesel (ULSD)

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: Water-accommodated fraction

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Water-accommodated fraction

Details on results:

- Behavioural abnormalities: Refer to tables with summarised test results
- Observations on body length and weight: Not applicable
- Other biological observations: None
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None

Results with reference substance (positive control):

Ultra low sulphur diesel (ULSD) was used as a reference gas oil which has good environmental claims and contains an antioxidant.
- Results with reference substance valid? Yes
- Mortality: Refer to tables with summarised test results
- EC50/LC50: Refer to tables with summarised test results

Reported statistics and error estimates:

Effects were investigated by visual inspection only. Now statistical procedures required.

Table 1: Summary table: Results of test with Daphnuia magna

Test Item	48 hour EL50 (mg/L)	NOEL (mg/L)
100% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear'	>1000	1000
76% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' : 24% ULSD	>1000	1000
50% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' : 50% ULSD	>1000	1000
24% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' : 76% ULSD	>1000	460
100% ULSD	100 - 300	100
Anullex BX	>1000	1000

Table: 2: Test results for 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear'

		Number of immobilised D. magna
Loading rate (mg/L)	Number of Daphnia magna	24 h	48 h
Control	10	0	0
	10	0	0
1000	10	0	0
	10	0	0

Table 3: Test results for ULSD

		Number of immobilised D. magna
Loading rate (mg/L)	Number of Daphnia magna	24 h	48 h
Control	10	0	0
	10	0	0
1	10	0	0
	10	0	0
3	10	0	0
	10	0	0
10	10	0	0
	10	0	0
30	10	0	0
	10	0	0
100	10	0	0
	10	0	0
300	10	3*	4*
	10	4*	8*
1000	10	10	10
	10	10	10

* daphnids slower moving than the control daphnids

Table 4: Test results for Annulex BX

		Number of immobilised D. magna
Loading rate (mg/L)	Number of Daphnia magna	24 h	48 h
Control	10	0	0
	10	0	0
1000	10	0	0
	10	0	0

Table 5: Test results for 24% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 76% ULSD

		Number of immobilised D. magna
Loading rate (mg/L)	Number of Daphnia magna	24 h	48 h
Control	10	0	0
	10	0	0
100	10	0	0
	10	0	0
220	10	0	0*
	10	0	0*
460	10	0*	0*
	10	0*	0*
1000	10	3*	3*
	10	4*	5*

* daphnids slower moving than the control daphnids

Table 6: Test results for 50% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 50% ULSD

		Number of immobilised D. magna
Loading rate (mg/L)	Number of Daphnia magna	24 h	48 h
Control	10	0	0
	10	0	0
100	10	0	0
	10	0	0
220	10	0	0
	10	0	0
460	10	0*	0
	10	0*	0
1000	10	0*	0
	10	0*	0

* daphnids slower moving than the control daphnids

Table 7: Test results for 76% 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' and 24% ULSD

		Number of immobilised D. magna
Loading rate (mg/L)	Number of Daphnia magna	24 h	48 h
Control	10	0	0
	10	0	0
100	10	0	0
	10	0	0
220	10	0	0
	10	0	0
460	10	0	0
	10	0	0
1000	10	0	0
	10	0	0

Validity criteria fulfilled:

yes

Conclusions:

The test result, expressed as an NOEC value, showed that the test material 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' was not acutely toxic at a loading rate of 1000 mg/L (nominal).
The test substance is non-toxic to D. magna. Using the European Union criteria for classifying ‘dangerous substances’ (EU, 1993), it is ‘not harmful’ to aquatic organisms.

Executive summary:

The acute toxicity of a sample of the substance 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' has been determined in tests with the freshwater invertebrate Daphnia magna (Water flea). The test was conducted in accordance with OECD Test Guidelines 202. 

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 (48h) 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. However in other respects they were considered to fulfil the requirements of current best practice.

The test results, expressed as the EL₅₀value, showed that the test material 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' was not acutely toxic to Daphnia magna at a loading rate of 1000 mg/L.

Using the European Union criteria for classifying ‘dangerous substances’ (EU, 1993), 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' would be classified as ‘not harmful’ to aquatic organisms.

The acute toxicity of 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' was determined both alone, and when blended with automotive gas oil (AGO) - ultra low sulphur diesel (ULSD). The toxicity of Anullex BX the antioxidant in AGO-ULSD was also assessed.

Reference 4

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: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C15 to C19

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. 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

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 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 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

- (48h) EL50 for Daphnia: >100 mg/l  [OECD 202; test mat. Distillates (Fischer–Tropsch), heavy, C18–C50 branched, cyclic and linear (WAFs) (nominal) based on: mobility];

- (48h) EL50 for Daphnia: >1000 mg/l  [OECD 202; test mat. Distillates (Fischer–Tropsch), C8–C26 branched and linear

(WAFs) (nominal) based on: mobility];

- (48h) EL50 for Daphnia: >100 mg/l  [OECD 202; test mat. Hydrocarbons, C15-C19, n-alkanes, isoalkanes < 2% aromatics (WAFs) (nominal) based on: mobility].

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 not available for 'Paraffin waxes (Fischer-Tropsch), full-range, C15-50 - branched and linear':

The short-term toxicity of the closely related substance GTL Base Oil Distillates (covering the carbon range from C18 to C50) 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 EL₅₀value, showed that the sample was not hazardous to Daphnia magna at a loading rate of 100 mg/l.

The acute toxicity of a sample of the supporting substance GTL Gasoil (covering the carbon range from C8 to C26) has been determined by Palmer (2001) in tests 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 EL₅₀value, showed that the sample was not acutely toxic to Daphnia magna at a loading rate of 1000 mg/l.

Albertus and Phillips (2005) have also reported results for an acute toxicity test carried out on the related substance GTL Gasoil (covering the carbon range from C8 to C26) 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.

Measured acute (short-term) toxicity data are available for the substance 'Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics' to the freshwater invertebrate Daphnia magna (Harris, 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.

Conclusion

Short-term Daphnia toxicity tests are available for three related substances in the relevant carbon number range. In the absence of adverse effects in these studies it can be concluded that, based on weight of evidence, the ELR₅₀(freshwater invertebrates) for 'Paraffin waxes (Fischer-Tropsch), full-range, C15-50 - branched and linear' is >100 mg/l.

It is safe to say that 'Paraffin waxes (Fischer-Tropsch), full-range, C15-50 - branched and linear' will not produce toxicity at the maximum attainable water solubility.