Naphtha (Fischer-Tropsch), light, C4-10-branched and linear

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 20 February 2011 and 10 March 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, 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 item in cases where the test item 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 item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 100 %
Batch number: not supplied
Storage conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Analysis of the WAF was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and the 100 mg/l loading rate WAF test group (replicates R1 - R4 pooled) at 0 and 48 hours (see Appendix 5 in any other information on results including tables section). Duplicate samples were taken and stored at approximately -20 degC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Range-finding test
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the range-finding test the test item was prepared as a Water Accommodated Fraction (WAF).

The loading rates to be used in the definitive test were determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/l.
Amounts of test item (23 and 230 mg) were each separately added to the surface of 2.3 litres of reconstituted water in mixing vessels with minimal headspace to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the reconstituted 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.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21°C to 22°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel was completely filled with test media and sealed with a ground glass stopper to prevent losses due to the volatile nature of the test item. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
The temperatures of the control and test cultures at 48 hours were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and the temperatures were within the test guideline specification.

Definitive test

Amounts of test item (23, 41, 74, 129 and 230 mg) were added separately to the surface of 2.3 litres of reconstituted water in separate stirring vessels with minimal headspace to give the loading rates of 10, 18, 32, 56 and 100 mg/l. After the addition of the test item, the reconstituted 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 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 48 hours (see Appendix 5).


- Controls:

A positive control (Harlan Laboratories Ltd., Project No: 41100002) 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, with the exception of the temperature which ranged from 18 to 22°C.

Analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977*) at 24 and 48 hours based on the nominal test concentrations gave the following results:

EC50(24 hrs) 1.6 mg/L 95% Confidence Limits 1.5-1.9 mg/L
EC50(48 hrs) 1.5mg/L 95% Confidence Limits 1.3-1.7 mg/L

The No Observed Effect Concentration after 24 and 48 hours was 1.0 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

* Hamilton, M A, Russo, R C and Thurston, R V (1977) Trimmed Spearman-Karber Method for Estimating Median Lethal Concentration in Toxicity Bioassays. Environ Sci Technol 11, 714-719.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Species
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 approximately 100 ml of Elendt M7 medium (see Appendix 1) 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 Water
The reconstituted water used for both the range-finding and definitive test is defined below.

Reconstituted Water-Elendt M7 Medium
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
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 uS cm"1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI 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 CaC03.

Macro Nutrient Stock Solutions

Solution Concentration of stock solution (g/l)
(I) CaCl2.2H2O 293.80
(II) NaHCO3 64.80
(III) MgSO4.7H2O 246.60
(IV) Na2SiO3.9H2O 50.00
(V) KCl 58.00
(VI) NaNO3 2.74
(VII) K2HPO4 1.84
(VIII) KH2PO4 1.43

Vitamin Nutrients

Solution Concentration of stock solution (mg/l)
(IX) Thiamine hydrochloride 750
Cyanocobalamine (vitamin
B12) 100
D(+) biotin (vitamin H) 75

The final medium was prepared by adding an aliquot of stock solution 2 along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water.
The pH of the prepared media was 8.0 ± 0.2 and stored at 21°C to 25°C.

Test type:

static

Water media type:

freshwater

Limit test:

no

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:

Water temperature was recorded daily throughout the test using a Hanna Instruments HI 93510 digital thermometer.

Temperature was maintained at 21degC to 22ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and the temperatures were within the test guideline specification.

The temperature measurements are given in Appendix 6 in any other information on results.

pH:

pH were recorded at the start and termination of the test. The pH was measured using a Hach HQ30d pH and dissolved oxygen meter.

The results of the pH measurements are given in Appendix 6 in any other information on results.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration were measured using a Hach HQ30d pH and dissolved oxygen meter.
The dissolved oxygen concentration measurements are given in Appendix 6 in any other information on results.

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/l.

Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.

Details on test conditions:

TEST SYSTEM
Exposure conditions
As in the range-finding test 250 ml glass conical flasks each completely filled with test preparation were used. At the start of the test 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then sealed with ground glass stoppers to reduce losses due to volatilisation and maintained in a temperature controlled room at 20°C to 22°C 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 item.
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.

The vortex depth was recorded at the start and end of the mixing period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 18 - < 32 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

10 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

Range-finding Test
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1, see any other information on results.
No immobilisation was observed at the test concentration of 10 mg/l loading rate WAF. However, immobilisation was observed at 100 mg/l loading rate WAF.
Based on this information loading rates of 10, 18, 32, 56 and 100 mg/l loading rate WAF were selected for the definitive test.

Definitive Test
Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (any other information on results). The relationship between percentage immobilisation and concentration at 48 hours is given in Figure 1 (see background material).
Visual inspection of the data at 24 and 48 hours gave the following results based on nominal loading rates:

24 hours EL50 >100 mg/L
48 hours EL50 18-32 mg/L

EL = Effective Loading Rate

It was not possible to determine an EL*50 value after 24 hours as no loading rate tested resulted in 50% immobilisation.
The No Observed Effect Concentrations after 24 and 48 hours exposure were 32 and 10 mg/l respectively. The No Observed Effect Concentration is based upon no significant immobilisation at this concentration.

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 (see Table 3 in any any further information on results).

Total organic carbon analysis
Samples of the control and all loading rate WAF's were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis (Appendix 5, any further information on results). 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 quantitation of the analytical method and hence did not provide definitive evidence of the presence of test item. This does not infer that no test item was in solution just that which was present was so at a concentration of less than 1.0 mg C/l.
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.

Results with reference substance (positive control):

- Results with reference substance valid?
Yes

Reported statistics and error estimates:

An estimate of the EL*50 values was given by inspection of the immobilisation data.
* EL = Effective Loading Rate

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
10	0	0
100	8	10

Table 2              Cumulative Immobilisation Data in the DefinitiveTest

NominalLoading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	1	0	1*	5
10	0	0	0	0	0	0	0	0
18	0	0	0	0	2	1	3	15
32	1	0	1	5	10	10	20	100
56	3	4	7	35	10	10	20	100
100	3	1	4	20	10	10	20	100

R1 - R2 = Replicates 1 and 2 * Single immobilised daphnid considered to be due to natural causes given that no further immobilisation occurred.

Table 3              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/I)
	Control		10		18
	*	+	*	+	*	+
Height of Water Column (cm)	12	12	20.5	20.5	20.5	20.5
Depth of Vortex (cm)	-0.2	-0.2	-0.2	-0.2	-0.2	-0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

	Nominal Loading Rate (mg/I)
	32		56		100
	*	+	*	+	*	+
Height of Media Column (cm)	19.0	19.0	19.0	19.0	20.5	20.5
Depth of Vortex (cm)	-0.2	-0.2	-0.2	-0.2	-0.2	-0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

*= Start of mixing period

+= End of mixing period

Appendix 4      

Validation of Mixing Period

Pre-study investigational 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 items, in the WAF. A WAF of a nominal loading rate of 100mg/l was prepared in duplicate in deionised reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. 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 aqueous phase was removed by siphon and samples taken for Total Organic Carbon analysis.

The results are summarised as follows:

Test Item Loading Rate (mg/l)	Time (Hours)
	24		96
	mg C/l	mg C/l Corrected for Control	mg C/l	mg C/l Corrected for Control
Control	<LOQ	-	<LOQ	-
100	4.41	4.41	4.00	4.00

LOQ = Limit of quantitation which was considered to be 1.0 mg C/l.

It is evident from this work that increasing the stirring period did not increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.

Appendix 5

Total Organic Carbon Analysis

1.        METHOD OF ANALYSIS

1.1      Summary

The concentration of Total Organic Carbon (TOC) in the 100 mg/l loading rate WAF was determined by Total Organic Carbon analysis.

1.2      Samples

Samples of the control and 100 mg/l loading rate WAF test group were taken for analysis at 0 (fresh media) and 48 hours (old media).

1.3      Standards

Standard solutions of potassium hydrogen phthalate (C8H5K04) were prepared in reverse osmosis water at concentrations of 10, 20, 50 and 100 mg Total Carbon/I. Standard solutions of sodium carbonate (Na2C03) were prepared in reverse osmosis water at concentrations of 5, 10, 20 and 33 mg Inorganic Carbon/I. These solutions were used for calibration of the carbon analyser.

1.4      Procedure

The standard and sample solutions were analysed for TOC using a Shimadzu TOC-VCPH high temperature Total Organic Carbon Analyser using the following conditions:

Total Carbon Channel
Temperature                       :          680ºC
Carrier gas                          :          zero grade air
Carrier gas flow rate          :         150 cc/min regulated at 50 psi
Catalyst                                :          platinum
Injection volume                 :          50 µl

Inorganic Carbon Channel
Temperature                       :          ambient
Carrier gas                          :          zero grade air
Carrier gas flow rate          :         150 cc/min regulated at 50 psi
Catalyst                                :          20% orthophosphoric acid
Injection volume                 :          50 µl

2.        RESULTS

Samples	Nominal Loading Rate (mg/l)	Concentration of TOC (mg C/l)	Concentration of TOC Corrected for Control (mg C/l)
0Hours (Fresh Media)	Control	2.43
	10	2.14	<Control
	18	1.82	<Control
	32	1.79	<Control
	56	1.80	<Control
	100	1.46	<Control
48Hours (Old Media)	Control	<LOQ
	10	<LOQ	<LOQ
	18	<LOQ	<LOQ
	32	<LOQ	<LOQ
	56	<LOQ	<LOQ
	100	<LOQ	<LOQ

LOQ = limit of quantitation which was considered to be 1.0 mg C/l.

Appendix 6      

Physico-Chemical Measurements

Nominal Loading Rate (mg/l)		0 hours				24 hours	48 hours
		pH	mg 02/l	%ASV*	T°C	T°C	pH	mg 02/l	%ASV*	T°C
Control	R1	8.0	8.8	97	20	22	8.3	8.2	94	22
	R2	8.0	8.6	95	20	22	8.2	8.1	93	22
10	R1	8.0	8.8	97	20	22	8.1	8.2	94	22
	R2	8.0	8.8	97	20	22	8.0	8.2	94	22
18	R1	7.9	8.9	98	20	22	8.0	8.3	95	22
	R2	7.9	8.9	98	20	22	8.0	8.3	95	22
32	R1	7.9	9.0	99	20	22	7.9	8.2	94	22
	R2	7.9	9.1	100	20	21	7.9	8.2	94	22
56	R1	7.9	8.8	97	20	22	7.9	8.0	92	22
	R2	7.9	8.7	96	20	21	7.9	8.1	93	22
100	R1	7.9	8.8	97	20	22	7.9	8.3	95	22
	R2	7.9	8.9	98	20	22	7.9	8.3	95	22

*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R1- R2 = Replicates 1 and 2

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 (Effective Loading Rate) value in the range of 18 - 32 mg/l loading rate WAF. The No Observed Effect Concentration at 48 hours was 10 mg/l loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method 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

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item at concentrations of 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of 20 to 22°C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

Results

The 48-Hour EL50 (Effective Loading Rate) for the test item to Daphnia magna based on nominal test concentrations was between 18 and 32 mg/l loading rate WAF. The No Observed Effect Concentration was 10 mg/l loading rate WAF.

Samples of the control and all loading rate WAF's 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 quantitation of the analytical method and hence did not provide definitive evidence of the presence of test item. This does not infer that no test item was in solution just that which was present was so at a concentration of less than 1.0 mg C/l.

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.

Description of key information

(48 h) EL50 for Daphnia magna (freshwater): > 18 < 32 mg/l (WAF, nominal, based on: immobilisation) [OECD 202; test mat. C4-C10 branched and linear hydrocarbons (light) – Naphtha]

Additional information

Short term toxicity to invertebrates by "Naphtha (Fischer Tropsch), light, C4 -C10 - branched and linear" was investigated according to OECD Guideline 202. The acute toxicity of the test item to the freshwater invertebrate Daphnia magna gave a 48-Hour EL50 (Effective Loading Rate) value in the range of 18 - 32 mg/l loading rate WAF based on immobilisation. The No Observed Effect Concentration at 48 hours was 10 mg/l loading rate WAF.