Hydrocarbons, C18-C24, iso-alkanes <2% aromatics

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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: Water-accommodated fraction

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: Water-accommodated fraction

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: Water-accommodated fraction

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Water-accommodated fraction

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

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was conducted between 27 November 2009 and 1 December 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, and at the request of the Sponsor, a preparation period of 72 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

Analytical monitoring:

yes

Details on sampling:

Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and each 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 Appendix 3 attached in overall remarks and attachments). Duplicate samples were taken and stored at approximately -20 DegC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

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 test concentrations 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). Following pre-study work, and at the request of the Sponsor, a preparation period of 72 hours was used.
Amounts of test item (110, 352 and 1100 mg) were each separately added to the surface of 11 litres of dechlorinated tap water to give the 10, 32 and 100 mg/l loading rates respectively. The stirring vessels were sealed with minimal headspace to reduce losses due to the possible volatile nature of the test item. 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 71 hours and the mixtures allowed to stand for 1 hour. Visual inspection of the 10, 32 and 100 mg/l WAFs showed cloudy water columns and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Parafilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Parafilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10, 32 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no particles or undissolved test item to be present.

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 Appendix 3 attached in overall remarks and attachments section).

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 approximately 20C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a 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 and additional experiment 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 Appendix 1 attached in overall remarks and attachments section.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not appliciable

Hardness:

The water hardness was observed to be in the range 140 to 170 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 Appendix 11 (attached in overall remarks and attachments section).

Test temperature:

The results of the physico-chemical measurements are given in Appendix 9 (attached in overall remarks and attachements section. Temperature was maintained at approximately 20 Deg C throughout the test.

pH:

There were no treatment related differences for pH.

Dissolved oxygen:

There were no treatment related differences for oxygen concentration. 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.

Salinity:

Not appliciable

Nominal and measured concentrations:

Total organic carbon analysis
Total Organic Carbon (TOC) analysis (see Appendix 3) 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 4.30 mg C/l. On Days 0, 5 and 16 the TOC analysis showed slightly high amounts of carbon in the test preparations which ranged from 5.45 to 10.84 mg C/l and the controls which ranged from 1.93 to 5.20 mg C/l. Analysis of the corresponding frozen duplicate samples showed the carbon concentration to range from 2.17 to 5.27 mg C/l (1.59 to 4.26 mg C/l for the control). It was therefore considered that the initial high results may have been possibly due to slight carry over from other samples during analysis. These values were considered not to affect the study.
TOC analysis of the old test media showed amount of carbon present to range from less than the LOQ to 2.13 mg C/l.
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.

Details on test conditions:

Exposure conditions
For each loading rate a single daphnid was placed in approximately 100 ml of the test preparation in 100 ml completely filled and sealed glass stoppered conical flasks to reduce losses of test item through volatility. For each test and control group ten replicate test vessels were prepared. The flasks were maintained at approximately 20DegC with a photoperiod of 16 hours light (427 to 496 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 to 10 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.

Additional Experiment
Due to the elevated TOC levels observed during the definitive test, an additional experiment was conducted using a single loading rate of 100 mg/l to compare any effects observed with those observed in the definitive test. This test was conducted following the same procedures used in the definitive test.

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 32 - < 100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 32 - < 100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

32 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

32 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Details on results:

All Appendices and Tables 1-8 are attached

Validation of Mixing Period
Pre-study investigational work (see Appendix 2) indicated that there was a significant increase in the amount of total organic carbon in the WAF when the preparation period was extended from 24 to 96 hours. At the request of the Sponsor a preparation period of 72 hours was used to maintain consistency with other studies conducted on the test item.

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 attached in overall remarks and attachments section. Data for each individual replicate flask are given in Appendices 5 to 8 attached in overall remarks and attachments section.
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).
O
bservations 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 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 71 hours stirring and a 1-Hour standing period the 10 to 100 mg/l loading rates were observed to be cloudy dispersions increasing in turbidity with increasing loading rates. Given that visually the 10 to 100 mg/l loading rates were observed to be dispersions it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed no particles of undissolved test item to be present. The freshly prepared 10, 32 and 100 mg/l loading rate WAFs were observed to be clear, colourless solutions whilst the old media were observed to be pale green dispersions due to the presence of algal cells used as feed for the daphnids.
Lethal Effects on the Parental Generation (P1)
Mortality (immobilisation) occurred predominantly at the highest loading rate of 100 mg/l resulting in 100% mortality by Day 14 indicating a prolonged toxic effect attributable to exposure of Daphnia magna to the test item.
Mortalities were also observed at the 10 and 32 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 10 and 32 mg/l loading rate WAF test groups were not significant (P0.05) when compared to the control group.

The following EL50*(immobilisation) values based on nominal loading rates were estimated at 24, 48 and 96 hours and Day 7 and calculated by the geometric mean method on days 14 and 21:
Time EL*50 (mg/l) 95% Cofidence Limits (mg/l)

24 hours >100 -
48 hours >100 -
96 hours >100 -
7 days approximately 50 -
14 days 57 32 - 100
21 days 57 32 - 100
There was a significant effect on size and colour of the daphnids in that surviving daphnids from Day 7 onwards at 100 mg/l loading rate WAF were markedly smaller and paler in colour than the control animals.
Sub-lethal Effects on the Parental Generation (P1)
After 21 days there were no statistically significant differences between the control and 10 and 32 mg/l loading rate WAF test groups in terms of the number of live young produced per adult (see Appendix 4). The 100 mg/l loading rate WAF test group data was not included in statistical analysis as exposure to the test item eliminated all the daphnids prior to Day 21 of the test.
The EL*50 (reproduction) value calculated by the geometric mean method on Day 21, based on nominal loading rates was 57 mg/l loading rate WAF with 95% confidence limits of 32 100 mg/l loading rate WAF.
After 21 days the length of each surviving adult was determined, the results of which are given in Appendix 4. The results showed that there were no statistically significant differences (P 0.05) between the control, 10 and 32 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 100 mg/l loading rate WAF test group data was not included in statistical analysis as exposure to the test item eliminated all the daphnids prior to Day 21 of the test.
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 the 10 and 32 mg/l loading rate WAF 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 9 of the test.
Due to the toxic effect of the test item, the parental generation (P1) of the 100 mg/l loading rate WAF test group were eliminated prior to the production of young.
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" was considered to be 100 mg/l loading rate WAF on the basis that at this loading rate, significant mortalities (immobilisation) were observed in the parental generation (P1) with all the daphnids being eliminated prior to the production of young.
No Observed Effect Loading Rate
The "No Observed Effect Loading rate" (NOEL) was 32 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

* EL = Effective loading rate
Additional Experiment
The environmental parameters for the additional test were comparable to those from the definitive test. Temperature was maintained at approximately 20°C throughout the test, air saturation values (ASV) ranged from 68% to 121%, the test the light intensity was observed to be in the range 445 to 504 lux and the water hardness was observed to be in the range 114 to 176 mg/l as CaCO3 in the control and the highest surviving test group throughout the test.
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 results of the additional experiment showed no significant immobilisation at the 100 mg/l loading rate WAF test concentration compared to the control over the 21-Day test period however, the adult daphnids in the 100 mg/l loading rate WAF test group were observed to be pale and significantly smaller (see Appendix 4 attached in overall remarks and attachements section) compared to the control.
Significantly fewer (P<0.05) young (see Appendix 4 attached in overall remarks and atatchements section) were produced by the 100 mg/l loading rate WAF test concentration compared to the control at the end of the 21-Day test period. Data for each individual replicate flask are given in Appendices 13 and 14 (attached in overall remarks and atttachments section.
TOC analysis (see Appendix 3 attached in overall remarks and attachements section) of the freshly prepared test preparations showed the amount of carbon present to range from less than the LOQ to 3.86 mg C/l whilst analysis of the old media showed the amount of carbon present to range from less than the LOQ to 1.90 mg C/l.
These additional results indicated that whilst the test item affected reproduction at the 100 mg/l loading rate WAF test concentration, the immobilisation in the adult daphnids observed in the definitive test may not have been a true effect of the test item. It should be noted that the test was conducted using a difficult substance, a mixture with components of differing solubilities, which led to difficulties in quantifying the test concentrations.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in significant mortalities at the loading rate of 100 mg/l resulting in 100% mortalities by Day 14.
The 21-Day EL*50 (immobilisation) value, based on nominal loading rates, for the parental Daphnia generation (P1) was calculated to be 57 mg/l loading rate WAF with 95% confidence limits of 32 - 100 mg/l loading rate WAF.
The 21-Day EL*50 (reproduction) based on nominal loading rates was 57 mg/l loading rate WAF with 95% confidence limits of 32 - 100 mg/l loading rate WAF.

The "Lowest Observed Effect loading rate" (LOEL) and the "No Observed Effect Loading rate" (NOEL) based on nominal loading rates were 100 and 32 mg/l loading rate WAF respectively.

The results of the additional experiment showed no significant immobilisation at the 100 mg/l loading rate WAF test concentration compared to the control over the 21-Day test period however, the adult daphnids in the 100 mg/l loading rate WAF test group were observed to be pale and significantly smaller compared to the control. In addition, significantly fewer (P<0.05) young were produced by the 100 mg/l loading rate WAF test concentration compared to the control at the end of the 21-Day test period.

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 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. Due to the possible volatile nature of the test item, the test was conducted using completely filled and sealed test vessels.

Results.

The 21-Day EL*₅₀(immobilisation) value, based on nominal loading rates, for the parental Daphnia generation (P₁) was calculated to be 57 mg/l loading rate WAF with 95% confidence limits of 32 - 100 mg/l loading rate WAF.

The 21-Day EL*₅₀(reproduction) value based on nominal loading rates was calculated to be 57 mg/l loading rate WAF with 95% confidence limits of 32 - 100 mg/l loading rate WAF.

The "Lowest Observed Effect Loading rate" was considered to be 100 mg/l loading rate WAF on the basis that at this loading rate, significant mortalities (immobilisation) were observed in the parental generation (P₁) with all the daphnids being eliminated prior to the production of young.

The "No Observed Effect Loading rate" was considered to be 32 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 (P³0.05) between the control and the 32 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 4.30 mg C/l. 

On Days 0, 5 and 16 the TOC analysis showed slightly high amounts of carbon in the test preparations which ranged from 5.45 to 10.84 mg C/l and the controls which ranged from 1.93 to 5.20 mg C/l. Analysis of the corresponding frozen duplicate samples showed the carbon concentration to range from 2.17 to 5.27 mg C/l (1.59 to 4.26 mg C/l for the control). 

It was therefore considered that the initial high results may have been possibly due to slight carry over from other samples during analysis. These values were considered not to affect the study.

TOC analysis of the old test media showed the amount of carbon present to range from less than the LOQ to 2.13 mg C/l. 

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.

Due to the elevated TOC levels observed during the definitive test, an additional experiment was conducted using a single loading rate of 100 mg/l to compare any effects observed with those observed in the definitive test. TOC analysis of the freshly prepared test preparations showed the amount of carbon present to range from less than the LOQ to 3.86 mg C/l whilst analysis of the old media showed the amount of carbon present to range from less than the LOQ to 1.90 mg C/l. No significant immobilisation was observed at the 100 mg/l loading rate WAF test concentration over the 21-Day test period however, the adult daphnids in the 100 mg/l loading rate WAF test group were observed to be pale and significantly smaller compared to the control.

Significantly fewer (P<0.05) young were produced by the 100 mg/l loading rate WAF test concentration compared to the control at the end of the 21-Day test period.

These addition results indicated that whilst the test item affected reproduction at the 100 mg/l loading rate WAF test concentration, the immobilisation in the adult daphnids observed in the definitive test may not have been a true effect of the test item. It should be noted that the test was conducted using a difficult substance, a mixture with components of differing solubilities, which led to difficulties in quantifying the test concentrations.

  CONCLUSION

Exposure ofDaphnia magnato the test item resulted in significant mortalities at the loading rate of 100 mg/l resulting in 100% mortalities by Day 14.

The 21-Day EL*₅₀(immobilisation) value, based on nominal loading rates, for the parental Daphnia generation (P₁) was calculated to be 57 mg/l loading rate WAF with 95% confidence limits of 32 - 100 mg/l loading rate WAF.

The 21-Day EL*₅₀(reproduction) based on nominal loading rates was 57 mg/l loading rate WAF with 95% confidence limits of 32 - 100 mg/l loading rate WAF.

The "Lowest Observed Effect loading rate" (LOEL) and the "No Observed Effect Loading rate" (NOEL) based on nominal loading rates were 100 and 32 mg/l loading rate WAF respectively.

The results of the additional experiment showed no significant immobilisation at the 100 mg/l loading rate WAF test concentration compared to the control over the 21-Day test period however, the adult daphnids in the 100 mg/l loading rate WAF test group were observed to be pale and significantly smaller compared to the control. In addition, significantly fewer (P<0.05) young were produced by the 100 mg/l loading rate WAF test concentration compared to the control at the end of the 21-Day test period.

*EL = Effective loading rate

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-09-03 to 2015-04-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study required for Chinese notification. Uncovered vessels.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

other: MEP, P.R. China the Guidelines for the Testing of Chemical No. 211 "Daphnia magna Reproduction Test" (Version 2, 2013).

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 1.0, 3.2, 10, 32 and 100 mg/l
- Sampling method: Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from in fresh and old media of the control and WAF of 100 mg/l twice a week. Duplicate samples were taken on day 0, 2, 5, 7, 12, 14 and 19 of the test.
The samples were filtered with 0.45 µm filter as soon as they are taken, the first 10 ml of filtrate were discarded and the following 10 ml were collected for TOC
determination. 2 drops of 2 N HCI were added into filtrate to make the pH value about equal to 2 and the filtrate was purged of the inorganic carbon with oxygen for 200 seconds to remove the inorganic carbon as C02 and then injected twice or once (at analysis day of 14 and 19) for TOC determination.
- Sample storage conditions before analysis: The filtrates were analyzed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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). The vortex depth was approximately 0.2 cm such that a dimple was formed at the water surface on each occasion. The preparation period was 24 hours.
Test solutions were prepared according to the properties of the test item. Amounts of the test item (0.01007-0.0109, 0.03205-0.0325, 0.1000-0.1009, 0.3200-0.3212 and 1.0003-1.0067g) were weighed respectively and added to 10 L of demineralized water in a glass bottle. The bottle with solution was put onto a magnetic stirrer and stirred (150-200 rpm) thoroughly for about 23 hours, and then allowed to stand for 1 hour. Then a flow device outlet was used to remove the aqueous phases or WAFs (the first 75-100 ml discarded) to give the test solutions. A microscopic inspection of WAFs was made; there were no globules of test item present for WAFs of September 10, 2014; but there were visible globules of test item present for WAFs of September 12, 2014 to September 29, 2014. So a glass wool filter plug (length of 3-5 cm) was placed in the flow device outlet to filter the WAFs from September 12, 2014 to September 29, 2014. The filtrated WAFs were inspected again to confirm no globules in WAFs.
The WAFs 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.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): A microscopic inspection of WAFs was made; there were no globules of test item present for WAFs of September 10, 2014; but there were visible globules of test item present for WAFs of September 12, 2014 to September 29, 2014. So a glass wool filter plug (length of 3-5 cm) was placed in the flow device outlet to filter the WAFs from September 12, 2014 to September 29, 2014. The filtrated WAFs were inspected again to confirm no globules in WAFs.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus
- Source: The stock animal was supplied by Applying Chemistry Department Pesticide Environmental Evaluation Lab of China Agricultural University.
- Age of parental stock (mean and range, SD): Less than 24 hours old. Not first blood progeny.
- Feeding during test
- Food type: living algal cells of Chlorella spp, Pseudokirchneriella subcapitata and Desmodesmus subspicatus. A concentrated algal suspension was fed to the Daphnia to minimise the volume of algal culture medium transferred to the test vessels.
- Amount and frequency: 0.1-0.2 mg C (organic carbon)/Daphnia/Day

ACCLIMATION
- Acclimation period: The stock animals were maintained in culture conditions (light, temperature, and medium) similar to those used in the
test.
- Acclimation conditions (same as test or not): Similar
- Type and amount of food: Not reported
- Feeding frequency: Not reported
- Health during acclimation (any mortality observed): Test animals were derived from a healthy stock (i.e. showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discoloured animals, etc.).

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The offspring produced by each parent animal were removed and counted daily from the appearance of the first brood to prevent them consuming food intended for the parent. The number of living offspring was counted, and the presence of dead offspring was also recorded.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

approximately 140 mg/l (as CaC03)

Test temperature:

Range 20.1 to 20.9°C

pH:

Range 7.30 - 8.50

Dissolved oxygen:

Range 6.24 to 9.34 mg/l

Nominal and measured concentrations:

Nominal: 0 (control), 1.0, 3.2, 10, 32 and 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): closed with foil to reduce evaporation and to avoid dust getting into the solutions.
- Material, size, headspace, fill volume: Glass, 200 ml or 250 ml filled with 100 mg/l test solution.
- Aeration: none reported
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): The test was performed with renewal of the test solutions three times a week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: about 100 ml water/per animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralized water treated with special softening technics. The water was aerated for at least 24 hours before use, to ensure dissolved oxygen saturation.
- Total organic carbon: 0.346 mg/l
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Salinity:
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH value, dissolved oxygen, water hardness and water temperature were determined once a week, in fresh and expired media, in the control and WAF of 100 mg/l. Temperature was monitored continuously in an additional vessel. The light intensity was measured at the start and the end of the test.
During the test, the concentration of test item as measured by TOC analysis was determined twice a week, in fresh and old media, in the control and WAFs of rates of 1.0, 3.2, 10, 32 and 100 mg/l.

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 16 h light/8 hr dark
- Light intensity: 400-800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The number of living offspring was counted, and the presence of dead offspring was also recorded daily.
Mortality among the parent animals was recorded daily.
The length of the parent animals (i.e. body length excluding the anal spine) was measured using a Motic K Series Stereo Microscope at the end of the test.
Other parameters that can be measured or calculated include: first brood time, juveniles of the first brood, total Juveniles of each parent, total broods of each parent, etc.

VEHICLE CONTROL PERFORMED: yes/no: n/a

RANGE-FINDING STUDY
- Information provided by the Sponsor showed that EL50 (EL: Effective Loading rate) was between in the range of 32-100 mg/l.

Reference substance (positive control):

not specified

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: the first brood time

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

32 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: the first brood time

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

32 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Total living offspring for each parent

Duration:

21 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: Total living offspring for each parent

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

32 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Body length of parent

Duration:

21 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: Body length of parent

Duration:

21 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: Parental mortality

Details on results:

- Mortality of parent animals: No significant mortality
- No. of offspring produced per day per female: Not reported
- Body length and weight of parent animals: Body length: 0.31 cm for the control, 0.30 cm for the 1.0, 3.2, 10 and 32 mg/l concentrations, and 0.24 mg/l for the 100 mg/l concentration.
- Type and number of morphological abnormalities: not reported
- Type and number of behavioural abnormalities: not reported
- Number of males and females (parental): not reported
- Time to first brood release or time to hatch: First brood: Control: 8.9 d; 1.0 mg/l: 7.9 d; 3.2 mg/l: 8 d; 10 mg/l: 8.9 d; 32 mg/l: 9.9 d; 100 mg/l: 10.5 d.
- Egg development time: Not reported
- Brood size: Not reported
- Time to sexual maturity: Not reported
- Type and magnitude of biochemical changes: Not reported
- Other biological observations: Not reported
- Effect concentrations exceeding solubility of substance in test medium: No

Table 1 Mean First Brood Time of Each Parent

Nominal Loading Rate (mg/l)	Mean±S.D.
	Mean First Brood Time for the10 replicates per concentration (day after exposure)	Mean Number born  or the10 replicates per concentration
Control	8.9± 1.29	12.2±4.76
1.0	7.9±0.88	18.6± 10.53
3.2	8±0.94	7.4±2.59
10	8.9±0.57	12.5±4.93
32	9.9±0.88	24.5±11.37
100	10.5± 1.18	9.5±7.01

Note: First brood time: It is the time when offspring were first observed in the test vessel

Table 2 Mean Parental Immobilisation and Mortality

Nominal Loading Rate (mg/l)	Mean parental mortality and immobilisation for the10 replicates per concentration (day after exposure)	Mean±S.D.
Control	10 % mortality	0.1 ±0.3
1.0	0 %	0.0±0.0
3.2	0 %	0.0±0.0
10	0 %	0.0±0.0
32	0 %	0.0±0.0
100	0 %	0.0±0.0

Table 3 Mean Number of Total Living Juveniles of Each Parent

Nominal Loading Rate (mg/l)	Mean number of juveniles per parent of the 10 replicates ±S.D.
Control	179±39
1.0	203±25
3.2	189±27
10	194±22
32	170±24
100	74±22

Table 4 Mean Body Length of Each Surviving Parent at the End of Test

Nominal Loading rate (mg/l)	Mean Body Length per Parent for the 10 replicates  ±S.D.
Control	0.31 ±0.02
1.0	0.30±0.01
3.2	0.30±0.02
10	0.30±0.02
32	0.30±0.01
100	0.24 ± 0.02

Validity criteria fulfilled:

yes

Conclusions:

'Distillates (Fischer-Tropsch), C8-26 - branched and linear', Daphnia magna Reproduction Test 21-day NOELR (No Observed Effect Loading Rate) value is 100 mg/l (nominal WAF concentration) (highest loading rate) based on mortality of parent, 10 mg/l based on first brood time, and 32 mg/l based on total living offspring for each parent and body length of parent.

Reference 4

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

1. Hypothesis for the analogue approach:
The hypothesis for the analogue approach is that the test substance GTL Base Oil Distillates (C18-C50 branched, cyclic and linear hydrocarbons – Distillates / 848301-69-9 / 482-220-0) is produced in the same Fischer-Tropsch process as GTL Gasoil which is the starting material from which the registration substance is produced by fractional distillation. The source substance contains constituents of the target substance, Hydrocarbons, C18-C24, iso-alkanes, <2% aromatics, although it covers a wider carbon number distribution. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). See Endpoint Summary (CSR Section 7.1.2.2 for additional information).
2. Source and target chemical(s)
The source substance GTL Base Oil Distillates (Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear) is composed of linear, branched and cyclic hydrocarbons of chain length C18-C50.
The source substance GTL Gasoil (C8-C26) (C8-C26 branched and linear hydrocarbons – Distillates) is the substance from which the registration substance is produced. GTL Gasoil is composed of linear, branched and cyclic hydrocarbons of chain length C8-C26.
The target substance, Hydrocarbons, C18-C24, iso-alkanes, <2% aromatics, is composed of linear, branched and cyclic hydrocarbons of chain length C18-24.
3. Analogue approach justification
The constituents of the source and target substances are all hydrocarbons. Identical constituents have identical toxicity profiles. There is no evidence of toxicity in the studies on the source substance, and no evidence of toxicity in studies on hydrocarbons with shorter carbon chain length and a narrower range of carbon number, therefore there is no evidence for mixture effects or for increased toxicity with increased concentration.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Description of key information

No studies are available for long-term toxicity to aquatic invertebrates with Hydrocarbons, C18-C24, isoalkanes, <2% aromatics, however, data are for two GTL-derived substances in the relevant carbon number range for the registered substance.

GTL Gasoil:

21-day EL50: 32-100 mg/l (test mat. (WAF) (nominal) based on survival and reproduction;

21-day LOELR: 100 mg/l (test mat. (WAFs) (nominal) based on: survival and reproduction;

21-day NOELR: 32 mg/l (test mat. (WAFs) (nominal) based on: survival and reproduction.

GTL Base oil distillates:

21-day NOELR: 100 mg/l (test mat. (WAFs) (nominal) based on: survival and reproduction.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

No studies are available for long-term toxicity to aquatic invertebrates with Hydrocarbons, C18-C24, isoalkanes, <2% aromatics, however, data are for two GTL-derived substances in the relevant carbon number range for the registered substance.

A Daphnia reproduction study has been conducted with GTL Gasoil using the freshwater species Daphnia magna (Goodband, 2011). The test was conducted in accordance with OECD 211 and was compliant with GLP.

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 test was conducted using sealed vessels to minimise any potential for volatile losses of the test substance.

The 21-day LOELR based on parental mortality was determined to be 100 mg/L load rate WAF, as significant mortalities (P≥ 0.05) were seen in the parental generation and all parental daphnids were eliminated prior to the production of young at this treatment level.

The 21-day NOELR, based on parental mortalities, was determined to be 32 mg/L loading rate WAF as no significant mortalities (P≥ 0.05) were observed in the parental generation and no significant differences were seen between the control and the 32 mg/L loading rate WAF test group in terms of numbers of live young produced per adult by day 21.  

There was no significant effect on time to first brood at any treatment level.

A supporting study conducted specifically for Chinese registration (Yang, 2015b) is also available with Daphnia magna OECD TG 211 (2012). Daphnids were exposed to water accommodated fractions (WAFs) of the test material over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days under semi-static test conditions.

The 21-day NOELR for parental mortality in the supporting study was determined to be 100 mg/L loading rate WAF, as no immobilisation was seen in the parental generation at any treatment level.  The 21-day NOELR for the number of live offspring produced was determined to be 32 mg/L as no significant differences were observed between the control and 32 mg/L WAF loading rate, but significant (P <0.001) differences were seen between 100 mg/L WAF loading rate and the control group.

The 21-day NOELR for body length was also determined to be 32 mg/L WAF loading rate as no significant differences were seen between individuals at this loading rate and the 32 mg/L control group, but significant (P <0.001) differences were seen at 100 mg/L WAF loading rate and its control group.  

The 21-day NOELR for time to first brood was determined to be 10 mg/L as there were no significant differences observed between the control and 10 mg/L WAF loading rates, significant differences (P <0.05) were seen at the 32mg/L WAF loading rate.

Justification for the GTL Gasoil Key Study with Daphnia magna

The results for GTL Gasoil in the key study (Goodband, 2011) and the supporting study (Yang, 2015b) are both considered reliable as they were performed using the appropriate OECD test guideline (OECD 211 TG).  Goodband (2011) is considered key because it was a fully GLP study and is given a reliability score corresponding to Klimisch 1, whilst the supporting study, which was conducted specifically for Chinese registration, complied with the Chinese State Environmental Protection Administration (SEPA 2004)   requirements rather than GLP and is given a reliability score corresponding to Klimisch 2.  

It is noted that in the supporting study, the 21-day NOELR for time until first brood sets the overall study NOELR at a WAF loading rate of 10 mg/l, whereas the key study found no effect on time to first brood and gave an overall study NOELR of 32 mg/l WAF loading rate.  As the quality of the supporting study data is considered less reliable than that of the key study, and the result does not trigger any changes in the environmental hazard classification, the key study value of 32 mg/L WAF loading rate is retained.

Discussion on methodology

In undertaking chronic toxicity testing the best use of all available data should be made. However, the assessment of ecotoxicity data for many petroleum products is complicated as products are UVBCs containing many individual substances with a range of solubilities. Chemical analyses of the aqueous concentrations of all constituents are not possible due to the complexity of the composition.

The concentration of each constituent dissolved in the water phase at any particular ‘loading’ should be maximized. The maximum possible water concentration of each component is typically achieved through prolonged stirring of the water-petroleum substance mixture to produce a Water Accommodated Fraction (WAF). WAFs are prepared individually and not by serial dilution of a single stock solution.  In addition, a sealed system approach may be appropriate for more volatile petroleum substances.

GTL Gasoil is a poorly water soluble UVBC and poses specific challenges when preparing aqueous solutions for toxicity testing.  This substance contains constituents with a range of physio-chemical properties (e.g. volatility, water solubility) and fall under the OECD guidance document 23 (2nd Edition) description and definition of “difficult to test” substances (Organisation for Economic Co-operation and Development [OECD] 2019). All long-term aquatic studies with GTL Gas oil followed the recommendations laid out for such substances.

GTL Base Oil Distillates

An invertebrate reproduction study has been conducted with GTL Base Oil Distillates using the freshwater species Daphnia magna (Goodband, 2010). The test was conducted in accordance with OECD 211 and was compliant with GLP.

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. 

Conclusion

Long-term invertebrate toxicity tests are available for two GTL-derived substances in the relevant carbon number range for the registered substance. In the test with GTL Base Oil Distillates (C18-C50) there were no adverse effects at the highest loading rate (100 mg/l). In the test with GTL Gasoil (C8-C26) the NOELR was determined to be 32 mg/l based on mortality in the parental generation at the highest rate. However, it is considered likely that the effects observed are attributable to the lower carbon number constituents of the GTL Gasoil. This is supported by measured data on constituents reported by CONCAWE in which long-term effects on Daphnia were observed with a C12 constituent, but no effects up to the limit of solubility were observed with constituents ≥C13 (see Section 7.1 of the Chemical Safety Report attached in Section 13). Based on weight of evidence it can therefore be concluded that for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics, the long-term NOELR for freshwater invertebrates is ≥100 mg/l.