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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 27 March 2011 and 8 April 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 and following a
1-Hour standing period, 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 (incl. QA statement)

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 WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) (see Appendix 3 in any other information on results including tables section). Duplicate samples were taken and stored at approximately -20ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Experimental preparation
An amount of test item (2300 mg) was added to the surface of 23 litres of dechlorinated tap water in a mixing vessel with minimal headspace to give the 100 mg/l loading rate. After the addition of the test item, the dechlorinated tap 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. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF.
This method of preparation was conducted in duplicate to give replicates Ri and R2.
Total Organic Carbon (TOC) analysis was performed on the test solutions at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) (see Appendix 3 in any other information on results including tables section).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test Species
The test was carried out using juvenile rainbow trout (Oncorhynchus my kiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 10 March 2011. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 23 March 2011 to 4 April 2011. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at approximately 14°C with a dissolved oxygen content of greater than or equal to 10.4 mg 02/l. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was zero mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.0 cm (sd = 0.1) and a mean weight of 0.83 g (sd = 0.06) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.29 g bodyweight/litre.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Hardness:

The test water used for the definitive test was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 1 attached in background material section.

Test temperature:

The test vessels were sealed and maintained at approximately 14ºC

The water temperature was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. Temperature was measured using Hanna Instruments HI 93510 digital thermometer.
Please see Physico-Chemical Measurements appendix 4 in any other information on results.

pH:

The pH concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH concentrations were measured using a Hach HQ30d pH and dissolved oxygen meter.
Please see Physico-Chemical Measurements appendix 4 in any other information on results.

Dissolved oxygen:

The dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The dissolved oxygen concentration were measured using a Hach HQ30d pH and dissolved oxygen meter .
Please see Physico-Chemical Measurements appendix 4 in any other information on results including tables.

Salinity:

No Data

Conductivity:

No Data

Nominal and measured concentrations:

Definitive test: 100 mg/l loading rate.

Details on test conditions:

Exposure conditions
In the definitive test 20 litre glass exposure vessels were used for each test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test was conducted using completely filled and sealed round bottomed glass exposure vessels due to the volatile nature of the test item. The test vessels were maintained at approximately 14°C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels received no auxiliary aeration. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item.
Data from the control group was shared with similar concurrent studies.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure test concentrations of soluble components of the test item were maintained over the test and to prevent the build up of nitrogenous waste products.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaC03. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 1 attached in background material section.

-Total Organic Carbon Analysis:
Please see Appendix 3 in any other information on results including tables section.

- Particulate matter:
Not measured

- Metals: Not Stated
- Pesticides: Not Stated
- Chlorine: Not Stated
- Alkalinity: Not Stated
- Ca/mg ratio: Not Stated
- Conductivity: Not Stated
- Culture medium different from test medium: Not Stated
- Intervals of water quality measurement: Not Stated


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST 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 mortalities or sub-lethal effects of exposure were observed.

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

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Range-finding Test
Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1 (see any other information on results). There were no sub-lethal effects of exposure during the range-finding test.
The results showed no mortalities at the 22, 46 and 100 mg/l loading rate WAFs.
Based on this information, a single loading rate, in duplicate, 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 mortalities or sub-lethal effects of exposure were observed.

Definitive Test: Mortality data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2 (see any other information on results) in any other infromation on materials and methods including tables section.
There were no mortalities in 14 fish exposed to a 100 mg/l loading rate WAF for a period of 96 hours. Inspection of the mortality data gave the following results:
Time (h) LL (Lethal Loading rate) 50 (mg/l)
3 >100
6 >100
24 >100
48 >100
72 >100
96 >100
The No Observed Effect Loading rate (NOEL) was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this loading rate.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Sub-lethal effects
There were no sub-lethal effects of exposure observed in 14 fish exposed to a 100 mg/l loading rate WAF for a period of 96 hours.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 4 (see any other information on results). Temperature was maintained at approximately 14°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Vortex depth measurements
The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion (Table 3, see any other information on results).

Observations on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start and end of each mixing period, and after the 1-Hour settlement period the 100 mg/l loading rate was observed to be a clear, colourless water column with an oily slick at the water surface. After siphoning and for the duration of the test, the 100 mg/l loading rate was observed to be a clear, colourless solution. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

Total organic carbon analysis
Samples of the control and 100 mg/l loading rate WAF were taken at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Analysis of the fresh media (Appendix 3, any other information on results) showed values of less than the limit of quantitation (LOQ) of the analytical method (assessed to be 1.0 mg C/l) to 0.12 mg C/l. Analysis of the old media showed values of less than the LOQ to 1.60 mg C/l. 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.
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.

Reported statistics and error estimates:

An estimate of the LL50 values was given by inspection of the mortality data.

Sublethal observations / clinical signs:

Table 1             Cumulative Mortality Data in the Range-finding test

Nominal Loading Rate (mg/l)	Cumulative Mortality (Initial Population= 3)
	3Hours	6Hours	24Hours	48Hours	72Hours	96Hours
Control	0	0	0	0	0	0
22	0	0	0	0	0	0
46	0	0	0	0	0	0
100	0	0	0	0	0	0

Table 2     Cumulative Mortalityin the DefinitiveTest

Nominal Loading Rate (mg/l)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
100 R1	0	0	0	0	0	0	0
100 R2	0	0	0	0	0	0	0

R₁– R₂= Replicates 1 and 2

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

FIRST MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	43.0	43.0	43.0	43.0	43.0	43.0
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

SECOND MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	44.0	44.0	44.0	44.0	45.0	45.0
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

THIRD MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	43.5	43.5	44.0	44.0	44.0	44.0
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

FOURTH MIXING PERIOD

	Nominal Loading Rate (mg/l)
	Control		100 R1		100 R2
	*	+	*	+	*	+
Height of Water Column (cm)	45.0	45.0	44.5	44 .5	44.5	44.5
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

R₁- R₂= Replicates 1 and 2

*= Start of mixing period

+= End of mixing period

Appendix2      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 100 mg/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 asessed to be 1.0 mg C/l.

Appendix3      Total Organic Carbon Analysis

1.        METHOD OF ANALYSIS

1.1      Summary

The concentration of Total Organic Carbon (TOC) in the WAF of the test item, was determined by Total Organic Carbon analysis.

1.2      Samples

Samples of the control and each replicate 100 mg/l loading rate WAF test group were taken for analysis at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 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)
0 Hours (Fresh Media)	Control	<LOQ	-
	100 R1	<LOQ	<LOQ
	100 R2	<LOQ	<LOQ
24 Hours (Old Media)	Control	<LOQ	-
	100 R1	1.60	1.60
	100 R2	1.15	1.15
24 Hours (Fresh Media)	Control	<LOQ	-
	100 R1	<LOQ	<LOQ
	100 R2	<LOQ	<LOQ
48 Hours (Old Media)	Control	<LOQ	-
	100 R1	<LOQ	<LOQ
	100 R2	<LOQ	<LOQ
48 Hours (Fresh Media)	Control	<LOQ	-
	100 R1	<LOQ	<LOQ
	100 R2	<LOQ	<LOQ
72 Hours (Old Media)	Control	2.36	-
	100 R1	1.98	< control
	100 R2	1.91	< control
72 Hours (Fresh Media)	Control	1.48	-
	100 R1	1.43	< control
	100 R2	1.60	0.12
96 Hours (Old Media)	Control	2.04	-
	100 R1	2.24	0.20
	100 R2	1.90	<Control

R₁– R₂= Replicates 1 and 2

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

Appendix4      Physico-Chemical Measurements

Nominal LoadingRate (mg/l)	0Hours (FreshMedia)				24Hours (OldMedia)				24Hours (FreshMedia)
	pH	mg 02/l	%ASV*	T°C	pH	mg 02/l	%ASV*	T°C	pH	mg 02/l	%ASV*	T°C
Control	7.5	10.3	102	15	7.5	6.7	66	15	7.5	10.4	103	15
100 Ri	7.5	10.1	100	15	7.4	7.4	73	15	7.5	10.5	104	15
100 R2	7.5	10.1	100	15	7.4	6.7	66	15	7.5	10.4	103	15

 

Nominal LoadingRate (mg/l)	48Hours (OldMedia)				48Hours (FreshMedia)				72Hours (OldMedia)
	pH	mg 02/l	%ASV*	T°C	pH	mg 02/l	%ASV*	T°C	pH	mg 02/l.	%ASV*	T°C
Control	7.4	8.4	83	15	7.5	10.7	106	15	7.7	8.1	80	15
100 Ri	7.6	8.6	85	15	7.5	10.6	105	15	7.6	8.5	84	15
100 R2	7.7	8.1	80	15	7.5	10.6	105	15	7.5	7.8	77	15

 

Nominal LoadingRate (mg/l)	72Hours (FreshMedia)				96Hours (OldMedia)
	pH	mg 02/l	%ASV*	T°C	pH	mg 02/l	%ASV*	T°C
Control	7.3	10.5	104	15	7.3	8.2	80	14
100 Ri	7.2	10.4	103	15	7.4	8.5	83	14
100 R2	7.3	10.6	105	15	7.4	8.2	80	14

*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 fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LL50 (Lethal Loading rate) value 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 item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test fish were exposed, in two groups of seven, to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for a period of 96 hours at a temperature of approximately 14°C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

The 96-Hour LL50 ( Lethal Loading rate) 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.

Samples of the control and 100 mg/l loading rate WAF were taken at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Analysis of the fresh media showed values of less than the limit of quantitation (LOQ) of the analytical method (assessed to be 1.0 mg C/l) to 0.12 mg C/l. Analysis of the old media showed values of less than the LOQ to 1.60 mg C/l.

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. 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

(96h) LL50 for freshwater fish rainbow trout (Oncorhynchus mykiss): >100 mg/l (WAF, nominal, based on: mortality) [OECD 203; test mat. C4-C10 branched and linear hydrocarbons (light) – Naphtha]

Additional information

The acute toxicity of GTL Naphtha to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated according to the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" and gave a 96-Hour LL50 (Lethal Loading rate) value of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.