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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 July 2011 and 16 August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction 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, 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: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Due to the low aqueous solubility and complex nature of the test item for the purpose of the definitive test the test item was prepared as a Water Accommodated Fraction (WAF).
Based on the results of an acute toxicity to 1st Instar Daphnia magna study (Harlan Laboratories Ltd, Project Number: 41005111) the following loading rates were assigned to the definitive test: 0.22, 0.70, 2.2, 7.0 and 22 mg/l.
Water samples were taken from the control and each surviving test group (replicates pooled) on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 (old and fresh media) and 21 (old media) for quantitative analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in Appendix 3.

Vehicle:

no

Details on test solutions:

Define test:
Based on the results of an acute toxicity to 1st Instar Daphnia magna study (Harlan Laboratories Ltd, Project Number: 41005111, see section 6.1.3) the following loading rates were assigned to the definitive test: 0.22, 0.70, 2.2, 7.0 and 22 mg/l.
Experimental Preparation:
Due to the low aqueous solubility and complex nature of the test item for the purpose of the definitive test the test item was prepared as a Water Accommodated Fraction (WAF).
Amounts of test item (15.75, 49.5, 157.5 and 495 mg) were each separately added, via a plastic syringe, to the surface of 22.5 litres of reconstituted water in separate stirring vessels with minimal headspace to give the 0.70, 2.2, 7.0 and 22 mg/l loading rates. A further amount of test item (4.95 mg) was added to a glass slide and suspended in the water column of 22.5 litres of reconstituted water in a stirring vessel with minimal headspace to give the 0.22 mg/l loading rate. 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 slight 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 Parafilm was submerged into each 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 Parafilm seal. The aqueous phases or WAFs were removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 0.22, 0.70, 2.2, 7.0 and 22 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 (old and fresh media) and 21 (old media) (see Appendix 3, attached in the background material).

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 reconstituted water (Elendt M7 medium) in a temperature controlled room at 21 °C to 22°C. A temperature at which the stock daphnids were kept was observed to be slightly in excess of the range given in the study plan of 20 ± 1°C. This deviation was considered not to have affected the outcome or the validity of the test as there was no effect on survival and reproduction of the stock cultures. 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 27 hours old. These young were removed from the cultures and used for testing. Some of the young used may have been up to approximately 27 hours old. This deviation was considered not to affect the integrity of the test as it was considered that the majority of the young used were less than 24 hours old. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not applicable

Hardness:

The water hardness of the control and the highest surviving test group in the fresh and old media was measured once per week. The water hardness was observed to be in the range 232 to 300 mg/l as CaC03 in the control and the highest surviving test group throughout the test. P

Test temperature:

Temperature of the test preparations was recorded daily throughout the test and before and after each test media renewal. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Measurements were made on one replicate for each loading rate. Temperature was maintained at 21 °C to 22 °C throughout the test. 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 that the temperatures were within the test guideline specification. hysico-Chemical Measurements (Recorded in One Replicate Test Vessel) of Oxygen (mg 02/l), Temperature (°C) and pH were given in Appendix 13.

pH:

pH was recorded before and after each test media renewal and measured using a Hach HQ30d Flexi Handheld meter. Measurements were made on one replicate for each loading rate. hysico-Chemical Measurements (Recorded in One Replicate Test Vessel) of Oxygen (mg 02/l), Temperature (°C) and pH were given in Appendix 13.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded before and after each test media renewal and measured using a Hach HQ30d Flexi Handheld meter. Measurements were made on one replicate for each loading rate.
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.
Physico-Chemical Measurements (Recorded in One Replicate Test Vessel) of Oxygen (mg 02/l), Temperature (°C) and pH were given in Appendix 13.

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Based on the results of an acute toxicity to 1st Instar Daphnia magna study (Harlan Laboratories Ltd, Project Number: 41005111, see section 6.1.3) the following loading rates were assigned to the definitive test: 0.22, 0.70, 2.2, 7.0 and 22 mg/l.
Due to the low aqueous solubility and complex nature of the test item for the purpose of the definitive test the test item was prepared as a Water Accommodated Fraction (WAF).

Details on test conditions:

Exposure conditions
For each loading rate a single daphnid was placed in 100 ml of the test preparation in 100 ml glass stoppered conical flasks. The test vessels were completely filled and sealed to prevent losses due to the volatile nature of the test item. For each test and control group ten replicate test vessels were prepared. The flasks were maintained at 21°C to 22°C with a photoperiod of 16 hours light (631 to 948 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. Each test vessel was randomly assigned to a position in the laboratory. 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 renewed 3 times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.
Each daphnid received approximately 2 to 5 pi of an algal suspension (Desmodesmus subspicatus) and approximately 14 to 28 pi of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
On a daily basis the numbers of live and dead of the "Parental" (Pi) generation, the numbers of live and dead "Filial" (Fi) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
The number of Daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks. At the end of the test, the length of each surviving parent animal was determined.

Physico-chemical measurements
Temperature of the test preparations and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Measurements were made on one replicate for each loading rate. The water hardness of the control and the highest surviving test group in the fresh and old media was measured once per week.

Chemical analysis at test loading rates
Water samples were taken from the control and each surviving test group (replicates pooled) on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 (old and fresh media) and 21 (old media) for quantitative analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in Appendix 3.

Evaluation of data
The EL50 (Effective Loading rate) (immobilisation) values were estimated by inspection of the data. The observed mortalities in the parental (Pi) generation of the 0.70, 2.2, 7.0 and 22 mg/l loading rate WAF test groups were compared to the control group using the corrected chi-squared statistic (Breslow and Day 1980).The EL50 (reproduction) value after 21 days was estimated by inspection of the data.
For the estimation of the "Lowest Observed Effect Loading rate" (LOEL) and the "No Observed Effect Loading rate" (NOEL) the numbers of live young produced per adult over the duration 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) (see Appendix 4). Results from the control and each test group Daphnia length data, determined for the surviving daphnids on termination of the test, 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) (see Appendix 4). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

> 22 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

22 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 22 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 22 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

RESULTS
Definitive Test
The observations for each test and control group are summarised in Tables 1 to 7 (see any further information on results). The total cumulative production of live young is given in Table 8 and the number of live young produced per adult is shown in Table 9, see any further information on results. The total number of offspring per adult daphnia (for each replicate) alive at the end of the test is shown in Figure 1 (attached as Figure in the background material). Data for each individual replicate flask are given in Appendices 5 to 10 (all attached in the background material).

Physico-chemical Measurements
The results of the physico-chemical measurements are given in Appendix 11 (attached background material). Temperature was maintained at 21 °C to 22°C 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 that the temperatures were within the test guideline specification.
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.
Throughout the test the light intensity was observed to be in the range 631 to 948 lux (see Appendix 12, attached background material).
The water hardness was observed to be in the range 232 to 300 mg/l as CaC03 in the control and the highest surviving test group throughout the test. The results of the water hardness measurements are given in Appendix 13 (attached background material).

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 and after 23 hours stirring and a 1-Hour standing period, the 0.22 mg/l loading rate was observed to be a clear, colourless water column with a glass slide suspended in the column whilst the 0.70, 2.2, 7.0 and 22 mg/l loading rates were observed to be clear, colourless water columns with oily globules of test item at the water surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning , and throughout the duration of the test the 0.22, 0.70, 2.2, 7.0 and 22 mg/l loading rates were observed to be clear, colourless solutions.

Validation Criteria
The following validation criteria were achieved during the test:
Required Actual

a Control mortality <20% 10%
b Dissolved oxygen > 3 mg O2/I > 7.4 mg 02/l
c pH (control group) deviation < 1.5 < 0.8
d Mean number of live young per > 60 after 21 days 100
surviving adult (control group)
e Coefficient of variation for < 25% 24%
control group


Lethal Effects on the Parental Generation (Pi)
Mortality was observed at the 0.70, 2.2, 7.0 and 22 mg/l loading rate WAF test groups. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortalities in the 0.70, 2.2, 7.0 and 22 mg/l loading rate WAF test groups were not significantly different (P>0.05) when compared to the control group.
No mortalities occurred at the 0.22 mg/l loading rate WAF test group throughout the test.
No further mortality occurred throughout the test and, hence, no prolonged effects attributable to exposure of Daphnia magna to the test item were observed.
The following EL*50 (immobilisation) values based on nominal loading rates were estimated throughout the test:

Time EL*50 (mg/l)
24 hours >22
48 hours >22
96 hours >22
7 days >22
14 days >22
21 days >22

The daphnids in all the loading rates were observed to be the same size and colour as the control animals.

Sub-lethal Effects on the Parental Generation (P1)
After 21 days there were no statistically significant differences between the control and all the test groups in terms of the number of live young produced per adult (see Appendix 4, attached background material).
The EL*50 (reproduction) value, based on nominal loading rates was estimated to be greater than 22 mg/l loading rate WAF.
After 21 days the length of each surviving adult was determined, the results of which are given in Appendix 4, attached background material). The results showed that there were no statistically significant differences (P> 0.05) between the control and all the test groups in terms of length of the daphnids after 21 days exposure to the test item (see Appendix 4, attached background material).

Effects on the Filial Generation (F1)
Information on the effects of the test item on the Fi generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.
Young were first produced in the control test group on Day 8 of the test.
Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

Lowest Observed Effect Loading Rate
The "Lowest Observed Effect Loading rate" (LOEL) was considered to be greater than 22 mg/l loading rate WAF on the basis that at this loading rate no significant mortalities (immobilisation) were observed in the parental generation (P1) and that there were no significant differences (P>0.05) between the control and the 22 mg/l loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

No Observed Effect Loading Rate
The "No Observed Effect Loading rate" (NOEL) was 22 mg/l loading rate WAF as there were no significant mortalities (immobilisation) observed in the parental generation (P1) and there were no significant differences (P>0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

Chemical Analysis of Test Loading Rates
The test item is a complex mixture. The results have been calculated based on the individual components of n-hexane, n-heptane, n-octane and n-nonane.
Analysis of the fresh media (see Appendix 3, attached background material) on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19 showed measured concentrations in the range of less than the limit of quantitation (LOQ) to 0.0695 mg/l were obtained for n-hexane and n-heptane, in the range of less than the LOQ to 0.0367 mg/l for n-octane and in the range of less than the LOQ to 0.00372 mg/l for n-nonane.
Analysis of the old media (see Appendix 3, attached background material) on Days 2, 5, 7, 9, 12, 14, 16, 19 and 21 showed measured concentrations in the range of less than the LOQ to 0.00392 mg/l were obtained for n-hexane, in the range of less than the LOQ to 0.00669 mg/l for n-heptane, in the range of less than the LOQ to 0.00723 mg/l for n-octane and less than the LOQ for n-nonane.
Given that 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.

Results with reference substance (positive control):

No positive control

Reported statistics and error estimates:

For the estimation of the "Lowest Observed Effect Loading rate" (LOEL) and the "No Observed Effect Loading rate" (NOEL) the numbers of live young produced per adult over the duration 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) (see Appendix 4, background material). Results from the control and each test group Daphnia length data, determined for the surviving daphnids on termination of the test, 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) (see Appendix 4, background material). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table 1: Summary of Findings Following the Exposure of Daphnia magna for 21 Days

Nominal Loading Rate(mg/l)	%Survival of P1	Number of Live Young		Number of Dead Young		Number of Unhatched Eggs
		Total	Per Female*(cumulative)	Total	Per Female (cumulative)	Total	Per Female (cumulative)
Control	90	904	100	0	0	0	0
0.22	100	1064	106	0	0	0	0
0.70	80	1031	110	0	0	0	0
2.2	80	925	97	0	0	0	0
7.0	80	997	101	1	<1	0	0
22	70	754	100	0	0	0	0

Table 2: Summary of Observations of the Control Group

Day	Adults Surviving	Number of Adults with Eggs in Brood Pouch	Live Young	Dead Young	Unhatched Eggs
1	10	0	0	0	0
2	10	0	0	0	0
3	10	0	0	0	0
4	10	0	0	0	0
5	10	3	0	0	0
6	10	9	0	0	0
7	10	10	0	0	0
8	9	8	67	0	0
9	9	9	20	0	0
10	9	9	13	0	0
11	9	9	122	0	0
12	9	9	29	0	0
13	9	9	71	0	0
14	9	9	123	0	0
15	9	9	45	0	0
16	9	9	95	0	0
17	9	9	98	0	0
18	9	9	51	0	0
19	9	9	78	0	0
20	9	9	74	0	0
21	9	9	18	0	0
		TOTALS	904	0	0

Table 3 Summary of Observations of the 0.22 mg/l Loading Rate WAF Test Group

Day	Adults Surviving	Number of Adults with Eggs in Brood Pouch	Live Young	Dead Young	Unhatched Eggs
1	10	0	0	0	0
2	10	0	0	0	0
3	10	0	0	0	0
4	10	0	0	0	0
5	10	0	0	0	0
6	10	9	0	0	0
7	10	10	0	0	0
8	10	10	76	0	0
9	10	10	20	0	0
10	10	10	13	0	0
11	10	10	157	0	0
12	10	10	2	0	0
13	10	10	23	0	0
14	10	10	265	0	0
15	10	10	0	0	0
16	10	10	19	0	0
17	10	10	219	0	0
18	10	10	0	0	0
19	10	10	28	0	0
20	10	10	229	0	0
21	10	10	13	0	0
		TOTALS	1064	0	0

Table 4 Summary of Observations of the 0.70 mg/l Loading Rate WAF Test Group

Day	Adults Surviving	Number of Adults with Eggs in Brood Pouch	Live Young	Dead Young	Unhatched Eggs
1	10	0	0	0	0
2	10	0	0	0	0
3	10	0	0	0	0
4	10	0	0	0	0
5	10	1	0	0	0
6	10	9	0	0	0
7	10	10	0	0	0
8	10	10	85	0	0
9	10	10	26	0	0
10	10	10	0	0	0
11	10	10	160	0	0
12	10	10	25	0	0
13	10	10	0	0	0
14	10	10	260	0	0
15	10	10	30	0	0
16	9	9	17	0	0
17	9	9	169	0	0
18	9	9	22	0	0
19	9	9	11	0	0
20	9	9	193	0	0
21	8	8	33	0	0
		TOTALS	1031	0	0

Table 5 Summary of Observations of the 2.2 mg/l Loading Rate WAF Test Group

Day	Adults Surviving	Number of Adults with Eggs in Brood Pouch	Live Young	Dead Young	Unhatched Eggs
1	10	0	0	0	0
2	10	0	0	0	0
3	10	0	0	0	0
4	10	0	0	0	0
5	10	3	0	0	0
6	10	8	0	0	0
7	10	9	0	0	0
8	10	10	65	0	0
9	10	10	0	0	0
10	10	10	24	0	0
11	10	10	118	0	0
12	10	10	24	0	0
13	10	10	63	0	0
14	10	9	176	0	0
15	10	10	26	0	0
16	10	10	57	0	0
17	10	10	140	0	0
18	10	0	0	0	0
19	9	9	65	0	0
20	9	9	134	0	0
21	8	8	33	0	0
		TOTALS	925	0	0

Table 6 Summary of Observations of the 7.0 mg/l Loading Rate WAF Test Group

Day	Adults Surviving	Number of Adults with Eggs in Brood Pouch	Live Young	Dead Young	Unhatched Eggs
1	10	0	0	0	0
2	10	0	0	0	0
3	10	0	0	0	0
4	10	0	0	0	0
5	10	0	0	0	0
6	10	6	0	0	0
7	10	9	0	0	0
8	10	9	50	0	0
9	10	10	21	0	0
10	10	10	0	0	0
11	10	10	102	0	0
12	10	10	118	0	0
13	10	10	11	0	0
14	10	10	142	0	0
15	10	10	140	0	0
16	10	10	34	0	0
17	10	10	106	0	0
18	9	9	21	0	0
19	9	9	102	1	0
20	8	8	95	0	0
21	8	8	55	0	0
		TOTALS	997	1	0

Table 7: Summary of Observations of the 22 mg/l Loading Rate WAF Test Group

Day	Adults Surviving	Number of Adults with Eggs in Brood Pouch	Live Young	Dead Young	Unhatched Eggs
1	10	0	0	0	0
2	10	0	0	0	0
3	9	0	0	0	0
4	9	0	0	0	0
5	9	1	0	0	0
6	9	7	0	0	0
7	9	9	0	0	0
8	9	9	47	0	0
9	9	9	9	0	0
10	9	9	10	0	0
11	9	9	101	0	0
12	9	9	55	0	0
13	9	9	0	0	0
14	7	7	178	0	0
15	7	7	29	0	0
16	7	7	0	0	0
17	7	7	142	0	0
18	7	7	0	0	0
19	7	7	27	0	0
20	7	7	129	0	0
21	7	6	27	0	0
		TOTALS	754	0	0

Table 8: Total Cumulative Production of Live Young

	Day
Nominal LoadingRate (mg/l)	1	2	3	4	5	6	7	8	9	10
Control	0	0	0	0	0	0	0	67	87	100
0.22	0	0	0	0	0	0	0	76	96	109
0.70	0	0	0	0	0	0	0	85	111	111
2.2	0	0	0	0	0	0	0	65	65	89
7.0	0	0	0	0	0	0	0	50	71	71
22	0	0	0	0	0	0	0	47	56	66

Table 8 continued

Nominal LoadingRate (mg/l)	Day
	11	12	13	14	15	16	17	18	19	20	21
Control	222	251	322	445	490	585	683	734	812	886	904
0.22	266	268	291	556	556	575	794	794	822	1051	1064
0.70	271	296	296	556	586	603	772	794	805	998	1031
2.2	207	231	294	470	496	553	693	693	758	892	925
7.0	173	291	302	444	584	618	724	745	847	942	997
22	167	222	222	400	429	429	571	571	598	727	754

Table 9 Number of Live Young Produced per Adult (Non-Cumulative)

Nominal LoadingRate (mg/l)	Day
	1	2	3	4	5	6	7	8	9	10
Control	0	0	0	0	0	0	0	7	2	1
0.22	0	0	0	0	0	0	0	8	2	1
0.70	0	0	0	0	0	0	0	9	3	0
2.2	0	0	0	0	0	0	0	7	0	2
7.0	0	0	0	0	0	0	0	5	2	0
22	0	0	0	0	0	0	0	5	1	1

Nominal LoadingRate (mg/l)
	Day
	11	12	13	14	15	16	17	18	19	20	21
Control	14	3	8	14	5	11	11	6	9.	8	2
0.22	16	0	2	27	0	2	22	0	3	23	1
0.70	16	3	0	26	3	2	19	2	1	21	4
2.2	12	2	6	18	3	6	14	0	7	15	4
7.0	10	12	1	14	14	3	11	2	11	11	7
22	11	6	0	20	4	0	20	0	4	18	4

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in no significant mortalities at all the loading rates employed during the test.
The 21-Day EL50 (immobilisation) value, based on nominal loading rates, for the parental Daphnia generation (P1) was estimated to be greater than 22 mg/l loading rate WAF.
No significant impairment of reproduction was observed at the loading rates employed during the test.
The 21-Day EL50 (reproduction) based on nominal loading rates was greater than 22 mg/l loading rate WAF.
The "Lowest Observed Effect Loading rate" (LOEL) based on nominal loading rates was considered to be greater than 22 mg/l loading rate WAF and the "No Observed Effect Loading rate" (NOEL) was considered to be 22 mg/l loading rate WAF.

EL = Effective Loading rate

Executive summary:

Introduction.

A study was performed to assess the effect of the test item on the reproduction of Daphnia magna over a 21-Day period. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 211 (2008) "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Methods.

Based on the results of an acute toxicity study (Harlan Laboratories Ltd., Project Number: 41005111), Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test item over a range of nominal loading rates of 0.22, 0.70, 2.2, 7.0 and 22 mg/l for a period of 21 days. WAFs were renewed 3 times per week. Due to the volatile nature of the test item, the test was conducted using completely filled and sealed test vessels. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.

Results.

The 21-Day EL50 (Effective Loading rate) (immobilisation) value, based on nominal loading rates, for the parental Daphnia generation (Pi) was estimated to be greater than 22 mg/l loading rate WAF. The 21-Day EL50 (reproduction) value based on nominal loading rates was estimated to be greater than 22 mg/l loading rate WAF. The "Lowest Observed Effect Loading rate" was considered to be greater than 22 mg/l loading rate WAF on the basis that at this loading rate no significant mortalities (immobilisation) were observed in the parental generation (P1) and that there were no significant differences (P>0.05) between the control and the 22 mg/l loading rate WAF test group in terms of numbers of live young produced per adult by Day 21. The "No Observed Effect Loading rate" was considered to be 22 mg/l loading rate WAF on the basis that at this loading rate there were no significant mortalities (immobilisation) observed in the parental generation (P1 and that there were no significant differences (P>0.05) between the control and the 22 mg/l loading rate WAF test group in terms of numbers of live young produced per adult by Day 21. The test item is a complex mixture. The results have been calculated based on the individual components of n-hexane, n-heptane, n-octane and n-nonane. Analysis of the fresh media on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19 showed measured concentrations in the range of less than the limit of quantitation (LOQ) to 0.0695 mg/l were obtained for n-hexane and n-heptane, in the range of less than the LOQ to 0.0367 mg/l for n-octane and in the range of less than the LOQ to 0.00372 mg/l for n-nonane. Analysis of the old media on Days 2, 5, 7, 9, 12, 14, 16, 19 and 21 showed measured concentrations in the range of less than the LOQ to 0.00392 mg/l were obtained for n-hexane, in the range of less than the LOQ to 0.00669 mg/l for n-heptane, in the range of less than the LOQ to 0.00723 mg/l for n-octane and less than the LOQ for n-nonane. Given that 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.

Description of key information

-(21 d) NOEL rate for Daphnia magna (freshwater): 22 mg/l (WAF, nominal, based on: immobilisation) [OECD 211; test mat. 'C4-C10 branched and linear hydrocarbons (light) – Naphtha]

-(21 d) EL50 for Daphnia magna (freshwater): > 22 mg/l (WAF, nominal, based on: immobilisation) [OECD 211; test mat. 'C4-C10 branched and linear hydrocarbons (light) – Naphtha]

-(21 d) EL50 for Daphnia magna (freshwater): > 22 mg/l (WAF, nominal, based on: reproduction) [OECD 211; test mat. 'C4-C10 branched and linear hydrocarbons (light) – Naphtha]

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

22 mg/L

Additional information

Long term toxicity to invertebrates was investigated according to OECD Guideline 211. Exposure of Daphnia magna to the test item resulted in no significant mortalities at all the loading rates employed during the test. The 21-Day EL50 (immobilisation) value, based on nominal loading rates, for the parental Daphnia generation (P1) was estimated to be greater than 22 mg/l loading rate WAF. No significant impairment of reproduction was observed at the loading rates employed during the test. The 21-Day EL50 (reproduction) based on nominal loading rates was greater than 22 mg/l loading rate WAF. The "Lowest Observed Effect Loading rate" (LOEL) based on nominal loading rates was considered to be greater than 22 mg/l loading rate WAF and the "No Observed Effect Loading rate" (NOEL) was considered to be 22 mg/l loading rate WAF.