Paraffin waxes (Fischer-Tropsch), isomerization

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:

The study was conducted between 27 November 2009 and 31 March 2010.

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 and OECD 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. Following pre-study work, a preparation period of 24 hours was used. 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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Total organic carbon analysis
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. 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) (see attachedAppendix 3). Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Details on test solutions:

Procedure
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 substances, in the WAF.
A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in dechlorinated tap water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis.

Definitive Test
Information provided by the Sponsor gave an EL/LL50 of greater than 100 mg/l. Therefore, as toxicity was not expected, the number of testconcentrations was reduced from five, as recommended in the Test Guideline, to three (10, 32 and 100 mg/l loading rate WAF). This was considered not to affect the validity of the study.

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). Based on pre-study work, a preparation period of 24 hours was used.
Amounts of test item (100, 320 and 1000 mg) were each separately added to the surface of 10 litres of dechlorinated tap water respectively to give the 10, 32 and 100 mg/l loading rates. 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 slight dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic inspection of the WAFs showed no micro-dispersions or globules of test item to be present. 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 10, 32 and 100 mg/l loading rate WAFs.
Total Organic Carbon (TOC) analysis was performed on the test preparations on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 (old and fresh media) and 21 (old media) (see attached Appendix 3).

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Species
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of dechlorinated tap water at a temperature of 18°C to 19°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test Water
The dechlorinated tap water used for the definitive test was the same as that used to maintain the stock animals.
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 CaCO3. 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 the attached Appendix 1.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not applicable.

Hardness:

The water hardness was observed to be in the range 128 to 188 mg/l as CaCO3 in the control and the highest surviving test group throughout the test.
The results of the water hardness measurements are given in the attached Appendix 11.

Test temperature:

Temperature was maintained at 20°C to 22°C throughout the test.

A single temperature during the test was measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered notto 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 results of the physico-chemical measurements are given in Appendix 9.

pH:

There were no treatment related differences for pH.

Dissolved oxygen:

There were no treatment related differences for oxygen concentration.

Salinity:

Freshwater used.

Details on test conditions:

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 the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digitalthermometer. 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.


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 in the control group.

Throughout the test the light intensity was observed to be in the range 410 to 452 lux ( please see attached Appendix 10).


Exposure conditions
For each loading rate a single daphnid was placed in 100 ml of the test preparation in 150 ml glass flasks which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The flasks were maintained at 20°C to 22°C with a photoperiod of 16 hours light (410 to 452 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 5 µl of a unicellular algal culture (Chlorella sp.) 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" (P1) generation, the numbers of live and dead "Filial" (F1) 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.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95 % CL not reported.

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

There were no statistically significant differences (P≥ 0.05) between the control and the 32 and 100 mg/l loading rate WAF groups in terms of length of daphnids.

Remarks on result:

other: 95 % CL not reported.

Duration:

21 d

Dose descriptor:

other: Lowest Observed Effect Loading rate" (LOEL)

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

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 100 mg/l loading rate WAF test group in terms of numbers of

Remarks on result:

other: 95 % CL not reported

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Validation of Mixing Period
Pre-study investigational work (see Appendix 2) indicated that there was no significant increase in the amount of total organic carbon in the WAF when the preparation period was extended for longer than 24 hours. Therefore the test was conducted using a
23-Hour stirring period followed by a 1-Hour settlement period.

Definitive Test
The observations for each test and control group are summarised in Tables 1 to 5. The total cumulative production of live young is given in Table 6 and the number of live young produced per adult is shown in Table 7. The total number of offspring per parent daphnia (for each replicate) alive at the end of the test is shown in Figure 1. Data for each individual replicate flask are given in Appendices 5 to 8.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion (see Table 8).

Observations on Test Material Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period the 10, 32 and 100 mg/l loading rates were observed to be clear, colourless water columns with oily globules of test item floating at the surface. After 23 hours stirring and a 1-Hour standing period the 10, 32 and 100 mg/l loading rates were observed to remain clear, colourless water columns with oily globules of test item floating at the surface. Microscopic inspection of the WAFs showed no micro-dispersions or globules of test item to be present. After siphoning and for the duration of the test, the 10, 32 and 100 mg/l loading rates were observed to be clear, colourless solutions.

Lethal Effects on the Parental Generation (P1)
Mortality was observed at the 10 mg/l loading rate WAF on Day 20. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortalities in the 10 mg/l loading rate WAF test group were not significant (P0.05) when compared to the control group.
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 >100
48 hours >100
96 hours >100
7 days >100
14 days >100
21 days >100
The daphnids in the 10, 32 and 100 mg/l loading rate WAF test groups 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).


The EL*50 (reproduction) value, based on nominal loading rates was estimated to be greater than 100 mg/l loading rate WAF.
After 21 days the length of each surviving adult was determined, the results of which are given in Appendix 12. The results showed that there were no statistically significant differences (P 0.05) between the control and the 32 and 100 mg/l loading rate WAF test groups in terms of length of the daphnids after 21 days exposure to the test item (see Appendix 4). The daphnids from the 10 mg/l loading rate WAF test group were shown to be statistically smaller (P<0.05) compared to the control group. A review of the data indicated that this was due to a few larger daphnids in the control group compared to the test groups. Given that no significant differences were observed between the control and remaining loading rate WAF test groups in terms of daphnia lengths, and that no significant differences were observed in terms of the number of live young produced between the control and all the test groups, it was considered that the difference observed was due to natural variation within the daphnia population rather than an effect of the test item.
* EL = Effective loading rate

Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 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 100 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 100 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 100 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.

Total organic carbon analysis
Total Organic Carbon (TOC) analysis of the freshly prepared test preparations (see Appendix 3) showed amount of carbon present within the 10, 32 and 100 mg/l loading rate WAF test vessels to range from less than the limit of quantitation (LOQ) for the method (1.0 mg carbon C/l) to 1.60 mg C/l. TOC analysis of the old test media showed the amount of carbon present to range from less than the LOQ to 1.20 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 methodand hence did not provide definitive evidence of stability of the test preparations.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Validation Criteria

The following validation criteria were achieved during the test:

	Required	Actual
a       Control mortality	≤20%	0%
b       Dissolved oxygen	> 3 mg O2/l	> 8.2mg O2/l
c       pH (control group)	deviation ≤ 1.5	0.9
d       Mean number of live young per surviving adult (control group)	≥ 60 after 21 days	64
e       Coefficient of variation for control group	≤ 25 %	13%

Please see attached table 1 to table 8.

Validity criteria fulfilled:

yes

Conclusions:

CONCLUSION
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) values, based on nominal loading rates, for the parental Daphnia generation (P1) were estimated to be greater than 100 mg/l loading rate WAF.
No significant impairment of reproduction was observed at the loading rates employed during the test.
The 21-Day EL*50 (reproduction) based on nominal loading rates was greater than 100 mg/l loading rate WAF.
The "Lowest Observed Effect Loading rate" (LOEL) based on nominal loading rates was considered to be greater than 100 mg/l loading rate WAF and the "No Observed Effect Loading rate" (NOEL) was considered to be greater than or equal to 100 mg/l loading rate WAF.

Executive summary:

Introduction.

A study was performed to assess the effect of the test item 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ on the reproduction of Daphnia magna over a 21-day period. The method followed that described in the OECD Guidelines for Testing of Chemicals No 211 (1998) "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Methods.

Based on data supplied by the Sponsor, Daphnia magna were exposed (10 replicates of a single daphnid per group) to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 32 and 100 mg/l for a period of 21 days. The WAFs were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension. 

Results.

The 21-Day EL*₅₀(immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (P₁) was estimated to be greater than 100 mg/l loading rate WAF.

The 21-Day EL*₅₀(reproduction) value based on nominal loading rates was estimated to be greater than 100 mg/l loading rate WAF.

The "Lowest Observed Effect Loading rate" was considered to be greater than 100 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 100 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 100 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 100 mg/l loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

Total Organic Carbon (TOC) analysis of the freshly prepared test preparations showed the amount of carbon present within the 10, 32 and 100 mg/l loading rate WAF test vessels to range from less than the limit of quantitation (LOQ) for the method (1.0 mg carbon C/l) to 1.60 mg C/l. TOC analysis of the old test media showed amount of carbon present to range from less than the LOQ to 1.20 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 stability of the test preparations.

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

CONCLUSION

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 EL^*₅₀(immobilisation) values, based on nominal loading rates, for the parental Daphnia generation (P₁) were estimated to be greater than 100 mg/l loading rate WAF.

No significant impairment of reproduction was observed at the loading rates employed during the test.

The 21-Day EL*₅₀(reproduction) based on nominal loading rates was greater than 100 mg/l loading rate WAF.

The "Lowest Observed Effect Loading rate" (LOEL) based on nominal loading rates was considered to be greater than 100 mg/l loading rate WAF and the "No Observed Effect Loading rate" (NOEL) was considered to be 100 mg/l loading rate WAF.

*EL = Effective loading rate

Description of key information

- NOELR (21 d): ≥100 mg/l [OECD 211; test mat. GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0); (WAFs) (nominal)];

- as hydrocarbons with carbon numbers >C50 are considered too hydrophobic and immobile to cause significant aquatic toxicity, the study results of this read-across substance (covering the entire low molecular weight fraction of the registration substance) are expected to represent the maximum achievable toxicity of the registration substance containing higher molecular weight constituents (C25-150, about 30-55 % >C50);

- chronic toxicity of the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' to aquatic invertebrates is considered to be no effects at the water solubility limit (saturation). 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Measured long-term toxicity data are not available for the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization'.
However, for the endpoint, reliable data of the closely related substance GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0) can be used, which is entirely appropriate to draw conclusions on the long-term toxicity of the registration substance to aquatic invertebrates.
The test item GTL base oil - consisting of linear and branched alkanes (paraffins) with a small amount of cycloalkanes in the carbon range from C18 to C50 - covers the complete low molecular weight fraction of the registration substance up to C50. Since hydrocarbons with carbon numbers >C50 are considered too hydrophobic and immobile to cause significant aquatic toxicity, the study results of this read-across substance are expected to represent the maximum achievable toxicity of the registration substance containing higher molecular weight constituents (C25-150, about 30-55 % >C50).

The Daphnia reproduction study with GTL base oil was conducted in accordance with OECD 211 and was compliant with GLP (Goodband, 2010). Daphnia magna were exposed to water accommodated fractions (WAFs) of the test material over a range of nominal loading rates of 10, 32 and 100 mg/l for a period of 21 days under semi-static test conditions. The WAFs were prepared by stirring for 23 hour and then leaving to stand for 1 hour.

The Lowest Observed Effect Loading Rate was considered to be greater than 100 mg/l on the basis that at this loading rate, no significant differences (P³0.05) were observed between the control and the 100 mg/l WAF loading rate in terms of numbers of live young produced per adult by Day 21.

The No Observed Effect Loading Rate (NOELR) was considered to be 100 mg/l 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 (P³0.05) between the control and the 100 mg/l loading rate WAF test group in terms of numbers of live young produced per adult by Day 21.

Total Organic Carbon (TOC) analysis of the freshly prepared test preparations showed amount of carbon present within the 10, 32 and 100 mg/l loading rate WAF test vessels to range from less than the limit of quantitation (LOQ) for the method (1.0 mg C/l) to 1.60 mg C/l. TOC analysis of the old test media showed amount of carbon present to range from less than the LOQ to 1.20 mg C/l. 

 

Based on these results,

• the long-term NOELR for freshwater invertebrates is considered to be >100 mg/l for the registration substance and


• the long-term toxicity to aquatic invertebrates is considered to be no effects at the water solubility limit (saturation).