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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 15 November 2011 and 12 January 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

InFish, Early-Life Stage Toxicity Test”, US Code of Federal Regulations, Title 40, Part 797, Section 1600.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity 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 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

Specific details on test material used for the study:

Purity: not applicable - complex mixture
Batch number: not supplied

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test item in the test preparations were verified by chemical analysis (replicates pooled) on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19, 21, 23, 26, 28, 30 (old and fresh media) and 33 (old media) (see Appendix 2 attached in background material section).

Water samples were taken from the control and all surviving test groups (replicates Ri and R2 pooled) on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19, 21, 23, 26, 28, 30 (fresh and old media) and 33 (old media) for quantitative analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
The method of analysis, recoveries and test preparation analysis are described in Appendix 2 attached in background material section.

Vehicle:

no

Details on test solutions:

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

Amounts of test item (23, 73.6, 230, 736 and 2300 mg) were each separately added to the surface of 23 litres of dechlorinated tap water in a mixing vessel with minimal headspace to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rates respectively. 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 mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessels, sealed end down, to a depth of approximately 5 cm from the bottom of the vessels. 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 WAFs were removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs.

The concentration and stability of the test item in the test preparations were verified by chemical analysis (replicates pooled) on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19, 21, 23, 26, 28, 30 (old and fresh media) and 33 (old media) (see Appendix 2, attached in the background material).

Test organisms (species):

Pimephales promelas

Details on test organisms:

The test was carried out using freshly laid eggs of fathead minnows (Pimephales promelas). The in-house breeding stock fish were bred at Harlan Laboratories Ltd and maintained in dechlorinated tap water in glass tanks with an activated carbon and biological filtration system.
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 25°C with a dissolved oxygen content of greater than or equal to
8.2 mg O2/I. The breeding stock fish were fed daily.
Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilised. Fertilised eggs were collected from the breeding tanks on 16 November 2011 and used for the definitive test. The eggs were less than 24 hours old on introduction into the test system.
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:

no

Total exposure duration:

33 d

Post exposure observation period:

n/a

Hardness:

The water hardness in each vessel was measured at the start and on termination of the test and was determined using the methods described in Fields and On-Site Test Methods for the Analysis of Waters (British Standards Institution 1993).
The water hardness values were observed to range from 138 to 156 mg/l as CaC03 at the start of the test and from 134 to 158 mg/l as CaC03 at termination of the test (see Appendix 6 attached in background material section.
The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Elga AC1) 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 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, water hardness values are given in Appendix 6, both attached in background material section.

Test temperature:

The water temperature was recorded daily throughout the test with the exception of Days 14 and 15 (old and fresh media) and Day 19 (old media) when the light intensity was not recorded due to instrument malfunction and on Day 23 when the light intensity was not recorded in error. This was considered not to affect the integrity of the test as the remainder of the readings indicated that the light intensity remained relatively consistent throughout the study. The measurements on Day 0, and after each test media renewal, represent those of the freshly prepared test concentrations while the measurements taken prior to each test media renewal and on termination of the test represent those of the used or 24-Hour old test preparations
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The temperature was also monitored approximately every hour in control replicate Ri using a Testo temperature logger with the exception of several hours where, due to a technical error, the data logger was not re-launched. This was considered not to affect the integrity of the test as no adverse effects were observed during the test.
Temperature was maintained at 25 ± 2°C throughout the test. The temperature measurements recorded in control replicate Ri throughout the test by a Testo temperature logger are presented in Figure 1. Some of the temperatures measured by the temperature logger were recorded to be slightly in excess of the 25 ± 2°C range given in the study plan. This was considered not to affect the results of the test as no adverse effects were observed during the test.

The results of the temperature measurements are given in Appendix 5 attached in background material section.

pH:

The pH was recorded daily throughout the test with the exception of Days 14 and 15 (old and fresh media) and Day 19 (old media) when the light intensity was not recorded due to instrument malfunction and on Day 23 when the light intensity was not recorded in error. This was considered not to affect the integrity of the test as the remainder of the readings indicated that the light intensity remained relatively consistent throughout the study. The measurements on Day 0, and after each test media renewal, represent those of the freshly prepared test concentrations while the measurements taken prior to each test media renewal and on termination of the test represent those of the used or 24-Hour old test preparations. The pH was measured using a Hach HQ30d Flexi Handheld meter. There were no treatment related differences for pH.

The results of the pH measurements are given in Appendix 5 attached in background material section.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded daily throughout the test with the exception of Days 14 and 15 (old and fresh media) and Day 19 (old media) when the light intensity was not recorded due to instrument malfunction and on Day 23 when the light intensity was not recorded in error. This was considered not to affect the integrity of the test as the remainder of the readings indicated that the light intensity remained relatively consistent throughout the study. The measurements on Day 0, and after each test media renewal, represent those of the freshly prepared test concentrations while the measurements taken prior to each test media renewal and on termination of the test represent those of the used or 24-Hour old test preparations. The dissolved oxygen concentration was measured using a Hach HQ30d Flexi Handheld meter. Dissolved oxygen content was greater than or equal to 8.2 mg O2/I.

The results of the dissolved oxygen concentrations are given in Appendix 5 attached in background material section.

Salinity:

n/a freshwater used

Nominal and measured concentrations:

Based on the results of an acute toxicity test (Harlan Laboratories Ltd., Project Number: 41005110R, see section 6.1.1) the following nominal loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/l. Duplicate test vessels were prepared from each loading rate WAF.

The test item contains a mixture of linear and branched alkanes. The test samples were monitored for concentrations of the representative n-alkanes; n-hexane, n-heptane, n-octane and n-nonane.
Chemical analysis of the fresh media test samples (see Appendix 2 attached in the background material) throughout the test showed measured concentrations of n-hexane to range from less than the limit of quantitation (LOQ) (assessed as 0.0025 mg/l) to 0.20 mg/l, n-heptane to range from less than the LOQ (assessed as 0.0026 mg/l) to 0.140 mg/l, n-octane to range from less than the LOQ (assessed as 0.0026 mg/l) to 0.0811 mg/l and n-nonane to range from less than the LOQ (assessed as 0.0026 mg/l) to 0.0216 mg/l.
Chemical analysis of the old media test samples (see Appendix 2 attached in the background material) throughout the test showed measured concentrations to be below or close to the LOQ for the representative n-alkanes.
The test item is known to be volatile, however under the conditions required to maintain the fish in a healthy state, it was not possible to conduct the test using sealed test vessels to minimise losses due to volatilisation.
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.

Details on test conditions:

For the definitive test glass exposure vessels containing 200 ml of test media were used for each control and test vessel. Two replicates were prepared for the control and each test concentration. From Day 14 onwards the test volume was increased to 2 litres due to the increased size of the larvae. At the start of the test 30 eggs were placed in each test vessel.
The number of fertilised eggs introduced per concentration was 60 ie. 30 per replicate.
The control group was maintained under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build-up of nitrogenous waste products.
The test vessels were maintained at 25 ± 2°C with a photoperiod of 16 hours light and
8 hours darkness with 20 minute dawn and dusk transition periods throughout the duration of the test. The eggs and larvae were not individually identified.
The start of hatching was observed on Day 4 of the test and completion of hatching on Day 5. The larvae were fed protozoan (Paramecia micronucleatum) only from Day 6 to Day 8 as the larvae were too small at this time to feed on brine shrimp nauplii. On Days
9 and 10 the larvae were fed protozoan (Paramecia micronucleatum) and brine shrimp nauplii as they were considered sufficiently large to eat brine shrimp nauplii. Both food types were fed on Days 9 and 10 to avoid a sudden change from one food source to another. On Day 11 and throughout the remainder of the test the larvae were fed brine shrimp nauplii only.
The number of dead eggs (up to completion of hatching), dead and live larvae and sublethal effects of exposure were recorded daily with the exception of the 3.2 mg/l loading rate WAF test group replicate Ri on Day 7 and the 32 mg/l loading rate WAF test group replicate Ri on Day 10 when the numbers were not recorded in error. This was considered not to affect the integrity of the test as, in each case, the following days assessment showed no additional mortalities. The criteria of death for eggs were marked loss of translucency and change in coloration leading to a white opaque appearance. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heart beat, white opaque coloration and lack of reaction to mechanical stimulus.

Light intensity was recorded daily throughout the test with the exception of Days 14 and 15 (old and fresh media) and Day 19 (old media) when the light intensity was not recorded due to instrument malfunction and on Day 23 when the light intensity was not recorded in error. This was considered not to affect the integrity of the test as the remainder of the readings indicated that the light intensity remained relatively consistent throughout the study.

Reference substance (positive control):

no

Duration:

33 d

Dose descriptor:

LOELR

Effect conc.:

> 100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: fish length and dry weight

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: fish length and dry weight

Details on results:

1. Observations
The number of dead eggs observed during the definitive test are given in Table 1 (see any other information on results). The number of dead larvae and hatched (live) larvae observed during the definitive test are given in Tables 2 and 3 respectively (see any other information on results). The number of hatched larvae (non-cumulative) per day are given in Table 4 (see any other information on results).
The number of dead eggs and larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed. The mean hatching success rate for the control group was 95% thereby satisfying the validation criterion of greater than 66% hatching rate. The mean hatching rates for the test groups ranged from 95% to 100% (Table 5, see any other information on results).
The mean survival rate of the larvae for the control group was 97% thereby satisfying the validation criterion for post-hatch survival success rate of greater than 70%. The mean survival rates for the test groups ranged between 99% and 100% (Table 5, see any other information on results).
The start of egg hatching was observed to be on Day 4 of the test and completion of hatching was observed on Day 5 of the test.
There were no significant mortalities observed in any of the test groups.

2. Sub-lethal Effects
There were no sub-lethal effects observed in the test.

3. Length and weight data
Statistical analysis of the length data and dry weight data by analysis of variance (see Appendix 3, attached in the background material) showed no significant differences (P>0.05) between the control and the 3.2, 10, 32 and 100 mg/l loading rate WAF test groups in terms of fish length or weight. For the 1.0 mg/l loading rate WAF, whilst there was no significant difference in terms offish weight compared to the control, statistical analysis indicated that the fish lengths were significantly reduced in the 1.0 mg/l loading rate WAF test group compared to the control. A review of the data indicated that this was due to a few small fish in the 1.0 mg/l loading rate WAF test group which skewed the data producing a statistical difference. Given that no significant differences in terms of fish length and weight were observed between the control and the 3.2, 10, 32 and 100 mg/l loading rate WAF test groups, it was considered that the observed difference between the control and 1.0 mg/l loading rate WAF was due to the nature of the data and not an effect of the test item. The fish length and dry weight data obtained at termination of the test are summarised in Table 6 (see any further information on results). Fish Length and Weight Values are given in Appendix IV, attached in the background material.
Given this information and data assessment above it was considered that no effect on survival or growth attributable to the test item was observed.

4. Lowest observed effect concentration
The "Lowest Observed Effect Loading Rate" (LOEL), based on nominal loading rates, was considered to be greater than 100 mg/l loading rate WAF on the basis that there were no significant decreases (P>0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

5. No observed effect concentration
The "No Observed Effect Loading Rate" (NOEL), based on nominal loading rates, was considered to be 100 mg/l loading rate WAF on the basis that there were no significant decreases (P>0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

6. Vortex depth measurements
The vortex depth was recorded at the start and end of each mixing period and was observed to produce a dimple at the water surface on each occasion and was observed to produce a dimple at the water surface.

7. 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 1.0, 3.2, 10, 32 and 100 mg/l loading rates were observed to be clear, colourless water columns with oily globules of test item at the water surface. After siphoning and for the duration of the test, the 1.0, 3.2, 10, 32 and 100 mg/l loading rates were observed to be a clear, colourless solutions. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

8. Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 5, attached in the background material. Temperature was maintained at 25 ± 2°C throughout the test, while there were no treatment related differences for oxygen concentration or pH. The temperature measurements recorded in control replicate Ri throughout the test by a Testo temperature logger are presented in Figure 1. Some of the temperatures measured by the temperature logger were recorded to be slightly in excess of the 25 ± 2°C range given in the study plan. This was considered not to affect the results of the test as no adverse effects were observed during the test.
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.
The water hardness values were observed to range from 138 to 156 mg/l as CaC03 at the start of the test and from 134 to 158 mg/l as CaC03 at termination of the test (see Appendix 6, attached in the background material).

9. Verification of test concentrations
The test item contains a mixture of linear and branched alkanes. The test samples were monitored for concentrations of the representative n-alkanes; n-hexane, n-heptane, n-octane and n-nonane.
Chemical analysis of the fresh media test samples (see Appendix 2, attached in the background material) throughout the test showed measured concentrations of n-hexane to range from less than the limit of quantitation (LOQ) (assessed as 0.0025 mg/l) to 0.20 mg/l, n-heptane to range from less than the LOQ (assessed as 0.0026 mg/l) to 0.140 mg/l, n-octane to range from less than the LOQ (assessed as 0.0026 mg/l) to 0.0811 mg/l and n-nonane to range from less than the LOQ (assessed as 0.0026 mg/l) to 0.0216 mg/l.
Chemical analysis of the old media test samples (see Appendix 2, attached in the background material) throughout the test showed measured concentrations to be below or close to the LOQ for the representative n-alkanes.

Results with reference substance (positive control):

not appliciable

Reported statistics and error estimates:

Evaluation of data
For the estimation of the "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) the length and dry weight data obtained on termination of the test for the control and each test group were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table1              Number of Dead Eggs in the Definitive Test 

NominalLoadingRate(mg/l)		Number of Dead Eggs (Initial Population= 30)
		Days					Total
		1	2	3	4	5 †
Control	R1	1	0	0	0	0	1
	R2	2	0	0	0	0	2
1.0	R1	2	0	0	0	0	2
	R2	1	0	0	0	0	1
3.2	R1	1	0	0	0	0	1
	R2	0	0	0	0	0	0
10	R1	0	0	0	0	0	0
	R2	1	0	0	0	0	0
32	R1	0	0	0	0	0	0
	R2	0	0	0	0	0	0
100	R1	1	0	0	0	0	0
	R2	0	0	0	0	0	0

†All surviving eggs hatched by day 5

R₁– R₂= Replicates 1 and 2

Table2              Number of Dead Larvae in the Definitive Test

Nominal Loading Rate (mg/l)	Number of Dead Larvae (Non-cumulative)
	Day
	0	1	2	3	4	5	6	7	8	9	10
Control           R1	0	0	0	0	0	0	1	0	0	0	0
R2	0	0	0	0	0	0	0	1	0	0	0
1.0     R1	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0	0
3.2     R1	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0	0
10      R1	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0	0
32      R1	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0	0
100     R1	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	1	0	0	0	0	0

R₁– R₂= Replicates 1 and 2

Table 2 (continued)          Number of Dead Larvae in the Definitive Test

Nominal Loading Rate (mg/l)	Number of Dead Larvae (Non-cumulative)
	Day
	11	12	13	14	15	16	17	18	19	20	21	22	23
Control         R1	0	0	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0	0	0	0
1.0        R1	0	0	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0	0	0	0
3.2          R1	0	0	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0	0	0	0
10          R1	0	0	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0	0	0	0
32          R1	0	0	0	0	0	0	0	0	0	1	0	0	0
R2	0	1	0	0	0	0	0	0	0	0	0	0	0
100         R1	0	0	0	0	0	0	0	0	0	0	0	0	0
R2	0	0	1	0	0	0	0	0	0	0	0	0	0

R₁– R₂= Replicates 1 and 2

Table 2 (continued)          Number of Dead Larvae in the Definitive Test

Nominal Loading Rate (mg/l)	Number of Dead Larvae (Non-cumulative)
	Day
	24	25	26	27	28	29	30	31	32	33
Control     R1	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0
1.0  R1	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0
3.2  R1	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0
10    R1	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0
32    R1	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0
100   R1	0	0	0	0	0	0	0	0	0	0
R2	0	0	0	0	0	0	0	0	0	0

R₁– R₂= Replicates 1 and 2

Table3              Cumulative Number of Hatched (Live) Larvae in the Definitive Test

Nominal Loading Rate (mg/l)	Number of Live Larvae (Cumulative)
	Day
	0	1	2	3	4	5	6	7	8	9	10
Control       R1	0	0	0	0	1	29	28	28	28	28	28
R2	0	0	0	0	0	28	28	27	27	27	27
1.0     R1	0	0	0	0	1	28	28	28	28	28	28
R2	0	0	0	0	1	29	29	29	29	29	29
3.2     R1	0	0	0	0	7	29	29	29	29	29	29
R2	0	0	0	0	0	30	30	30	30	30	30
10        R1	0	0	0	0	1	30	30	30	30	30	30
R2	0	0	0	0	0	29	29	29	29	29	29
32        R1	0	0	0	0	1	30	30	30	30	30	30
R2	0	0	0	0	0	30	30	30	30	30	30
100        R1	0	0	0	0	0	29	29	29	29	29	29
R2	0	0	0	0	0	29	29	29	29	29	29

R₁– R₂= Replicates 1 and 2

Table 3 (continued)          Cumulative Number of Hatched (Live) Larvae in the Definitive Test

Nominal Loading Rate (mg/l)	Number of Live Larvae (Cumulative)
	Day
	11	12	13	14	15	16	17	18	19	20	21	22	23
Control         R1	28	28	28	28	28	28	28	28	28	28	28	28	28
R2	27	27	27	27	27	27	27	27	27	27	27	27	27
1.0          R1	28	28	28	28	28	28	28	28	28	28	28	28	28
R2	29	29	29	29	29	29	29	29	29	29	29	29	29
3.2          R1	29	29	29	29	29	29	29	29	29	29	29	29	29
R2	30	30	30	30	30	30	30	30	30	30	30	30	30
10             R1	30	29	29	29	29	29	29	29	29	29	29	29	29
R2	29	29	29	29	29	29	29	29	29	29	29	29	29
32             R1	30	30	30	30	30	30	30	30	30	30	30	30	30
R2	30	30	30	30	30	30	30	30	30	30	30	30	30
100             R1	29	29	29	29	29	29	29	29	29	29	29	29	29
R2	29	29	28	28	28	28	28	28	28	28	28	28	28

R₁– R₂= Replicates 1 and 2

Table 3 (continued)          Cumulative Number of Hatched (Live) Larvae in the Definitive Test

Nominal Loading Rate (mg/l)	Number of Live Larvae (Cumulative)
	Day
	24	25	26	27	28	29	30	31	32	33
Control        R1	28	28	28	28	28	28	28	28	28	28
R2	27	27	27	27	27	27	27	27	27	27
1.0     R1	28	28	28	28	28	28	28	28	28	28
R2	29	29	29	29	29	29	29	29	29	29
3.2     R1	29	29	29	29	29	29	29	29	29	29
R2	30	30	30	30	30	30	30	30	30	30
10        R1	29	29	29	29	29	29	29	29	29	29
R2	29	29	29	29	29	29	29	29	29	29
32        R1	30	30	30	30	30	30	30	30	30	30
R2	30	30	30	30	30	30	30	30	30	30
100        R1	29	29	29	29	29	29	29	29	29	29
R2	29	29	29	29	29	29	29	29	29	29

R₁– R₂= Replicates 1 and 2

Table4              Number of Hatched (Live) Larvae (Non-cumulative) in the Definitive Test

Nominal Loading Rate (mg/l)	Number of Hatched Larvae (Non-cumulative)
	Days
	1	2	3	4	5  †	Total
Control       R1	0	0	0	1	28	29
R2	0	0	0	0	28	28
1.0          R1	0	0	0	1	27	28
R2	0	0	0	1	28	29
3.2          R1	0	0	0	7	22	29
R2	0	0	0	0	30	30
10             R1	0	0	0	1	29	30
R2	0	0	0	0	29	29
32             R1	0	0	0	1	29	30
R2	0	0	0	0	30	30
100             R1	0	0	0	0	29	29
R2	0	0	0	0	29	29

†All surviving eggs hatched by day 5

R₁– R₂= Replicates 1 and 2

 Table5              Hatching and Survival Rates in the Definitive Test

Nominal Loading Rate (mg/l)	Hatching Rate (%)	Mean Hatching Rate (%)	Survival Rate (%)	Mean Survival Rate (%)
Control      R1	97	95	97	97
R2	93		96
1.0       R1	93	95	100	100
R2	97		100
3.2       R1	97	99	100	100
R2	100		100
10         R1	100	99	97	99
R2	97		100
32        R1	100	100	100	100
R2	100		100
100       R1	97	97	100	99
R2	97		97

R₁– R₂= Replicates 1 and 2

Table 6: Summary of fish length and dry weight

	Nominal Loading Rate (mg/l)
	Control	1.0	3.2	10	32	100
Body Length (mean ± standard deviation, mm)	14.96 ± 0.96	14.42 ± 1.17	14.70 ± 0.92	15.15 ± 0.99	14.94 ± 0.93	15.20 ± 0.84
Dry Weight (mean ± standard deviation, mg)	7.9 ± 1.8	7.2 ± 2.0	7.5 ± 1.4	8.1 ± 1.7	7.9 ± 1.7	8.4 ± 1.6

Validity criteria fulfilled:

yes

Conclusions:

The application of the test item to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae. The No Observed Effect Loading Rate was considered to be 100 mg/l loading rate WAF.

Executive summary:

Introduction.

A study was performed to assess the effects of the test item on freshly hatched larvae of the fathead minnow (Pimephales promelas). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 210, "Fish, Early-Life Stage Toxicity Test", US Code of Federal Regulations, Title 40, Part 797, Section 1600 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.1400.

Methods.

Based on the results on an acute toxicity test (Harlan Laboratories Ltd., Project Number: 41005110), newly laid eggs were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/l for a period of 33 days at a temperature of 25 ± 2°C under semi-static test conditions. The number of mortalities or any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and dry weight of the surviving fish were measured.

Results.

The test item contains a mixture of linear and branched alkanes. The test samples were monitored for concentrations of the representative n-alkanes; n-hexane, n-heptane, n-octane and n-nonane. Chemical analysis of the fresh media test samples throughout the test showed measured concentrations of n-hexane to range from less than the limit of quantitation (LOQ) (assessed as 0.0025 mg/l) to 0.20 mg/l, n-heptane to range from less than the LOQ (assessed as 0.0026 mg/l) to 0.140 mg/l, n-octane to range from less than the LOQ (assessed as 0.0026 mg/l) to 0.0811 mg/l and n-nonane to range from less than the LOQ (assessed as 0.0026 mg/l) to 0.0216 mg/l. Chemical analysis of the old media test samples throughout the test showed measured concentrations to be below or close to the LOQ for the representative n-alkanes. The test item is known to be volatile, however under the conditions required to maintain the fish in a healthy state, it was not possible to conduct the test using sealed test vessels to minimise losses due to volatilisation.

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. Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of fathead minnow (Pimephales promelas) larvae to 1.0, 3.2, 10, 32 and 100 mg/l loading rate WAFs. The mean hatching rate ranged from 95% to 100% and the mean survival rate ranged from 97% to 100%. The fish length and dry weight data obtained at termination of the test are summarised as follows:

	Nominal Loading Rate (mg/l)
	Control	1.0	3.2	10	32	100
Body Length (mean ± standard deviation, mm)	14.96 ± 0.96	14.42 ± 1.17	14.70 ± 0.92	15.15 ± 0.99	14.94 ± 0.93	15.20 ± 0.84
Dry Weight (mean ± standard deviation, mg)	7.9 ± 1.8	7.2 ± 2.0	7.5 ± 1.4	8.1 ± 1.7	7.9 ± 1.7	8.4 ± 1.6

Statistical analysis of these data showed there were no significant decreases (P>0.05) between the control and the 3.2, 10, 32 and 100 mg/l loading rate WAF test groups in terms of fish length or weight. For the 1.0 mg/l loading rate WAF, whilst there was no significant difference in terms of fish weight compared to the control, statistical analysis indicated that the fish lengths were significantly reduced in the 1.0 mg/l loading rate WAF test group compared to the control. A review of the data indicated that this was due to a few small fish in the 1.0 mg/l loading rate WAF test group which skewed the data producing a statistical difference. Given that no significant differences in terms of fish length and weight were observed between the control and the 3.2, 10, 32 and 100 mg/l loading rate WAF test groups, it was considered that the observed difference between the control and 1.0 mg/l loading rate WAF was due to the nature of the data and not an effect of the test item.

Conclusion.

Given the above results and information it was considered that the test item had no effect on the survival or growth of newly laid eggs of fathead minnows. The "Lowest Observed Effect Loading Rate" (LOEL), based on nominal loading rates, was considered to be greater than 100 mg/l loading rate WAF on the basis that there were no significant decreases (P>0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

The "No Observed Effect Loading Rate" (NOEL), based on nominal loading rates, was considered to be 100 mg/l loading rate WAF on the basis that there were no significant decreases (P>0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

Description of key information

(33 d) NOEL rate for fathead minnow (Pimephales promelas): 100 mg/l (WAF, nominal, based on: fish length and dry weight) [OECD 210; test mat. C4-C10 branched and linear hydrocarbons (light) – Naphtha]

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

Long-term toxicity to fish was investigated according to OECD Test Guideline 210. It was considered that the test item had no effect on the survival or growth of newly laid eggs of fathead minnows. The "Lowest Observed Effect Loading Rate" (LOEL), based on nominal loading rates, was considered to be greater than 100 mg/l loading rate WAF on the basis that there were no significant decreases (P>0.05) in terms of fish length and dry weight when compared to the control at the end of the test. The "No Observed Effect Loading Rate" (NOEL), based on nominal loading rates, was considered to be 100 mg/l loading rate WAF on the basis that there were no significant decreases (P>0.05) in terms of fish length and dry weight when compared to the control at the end of the test.