[No public or meaningful name is available]

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 16 October 2009 and 22 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not Applicable

Analytical monitoring:

yes

Details on sampling:

Range-finding test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Chironomus riparius larvae were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/kg (dry weight of sediment).

Definitive test
Based on the results of the range-finding test the following concentrations were assigned to the definitive test: 100, 180, 320, 560 and 1000 mg/kg (dry weight of sediment).

Vehicle:

no

Details on sediment and application:

Formulated Sediment
A defined formulated sediment was used with the following composition:
Industrial quartz sand 76% w/w
Kaolinite clay 20% w/w
Sphagnum moss peat 4% w/w

The peat was air dried and homogenised to give a particle size of less than 1 mm. The organic carbon content of the final mixture was 1.9%.


Amounts of test item (5.0, 50 and 500 mg) were each separately added to 380 g of quartz sand in a mixing vessel and mixed in a cement mixer for approximately 24 hours after which 100 g of clay and 20 g of air dried peat were added to each mixing vessel and mixed using a cement mixer for a further approximate 24 hours. An aliquot (196 ml) of deionised reverse osmosis water was added to each prepared sediment and mixed using a Kenwood Chef mixer to give the 10, 100 and 1000 mg/kg test concentrations respectively with nominal moisture content of approximately 40% of dry weight. The pH of each prepared sediment was adjusted to 7.0 ± 0.5 with calcium carbonate. The control was prepared in a similar manner, without the addition of test item.
Each prepared sediment was dispensed to a 600 ml glass beaker to give a 2 cm layer and was then covered with a 8 cm depth of reconstituted water (sediment:water ratio, 1:4). A plastic disc was placed over the sediment and the reconstituted water poured gently onto the surface of the disc in order to avoid disturbance of the sediment. The disc was removed after addition of the water. Two replicates were prepared for the control and each test concentration. This was a deviation to the study plan which stated that the range-finding test would be conducted using single replicates. This was considered not to have affected the validity of the test. The test vessels were then aerated (approximately 1 bubble/second) via narrow bore glass tubes approximately
2 – 3 cm above the sediment layer and the vessels left for seven days prior to addition of the test organisms in order to allow settlement and equilibration of test concentrations between the sediment and water phases.

After the 7-day equilibration period the aeration was stopped and 20 larvae were placed in each replicate test and control vessel and maintained in a temperature controlled room at 21ºC to 23ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. Some of the room temperatures were observed to be slightly in excess of the range given in the study plan of 20 ± 2°C. This deviation was considered not to have affected the outcome or the validity of the test as no adverse effects were observed in the control group throughout the test.

The aeration was switched back on after approximately 24 hours having allowed the larvae to settle in the sediment.

The larvae were fed at a rate of 0.25 to 0.5 mg Tetramin® flake food per larva per day for the first 10 days and 0.5 to 1.0 mg Tetramin® flake food per larva per day thereafter. The Tetramin® flake food was prepared as a suspension and an appropriate volume added to the overlying water.
The measured end-point for the test was the number of live, emerged adult midges. The number of emerged adult midges was recorded daily until termination of the test after 28 days. The sex of the individual midges was also determined after emergence. The male midges were identified by the presence of plumose antennae and the female by the absence of plumose antennae.

The control group was maintained under identical conditions but not exposed to the test item.

Data from the control group was shared with similar concurrent studies.

Test organisms (species):

Chironomus riparius

Details on test organisms:

The test was carried out using larvae (2 – 3 days old) of Chironomus riparius derived from in-house laboratory cultures.
Larvae were maintained in glass crystalline dishes with a 10-20 mm layer of fine quartz sand covered by reconstituted water (Elendt M4) in a temperature controlled room at 21ºC to 24ºC. The lighting cycle was controlled to give a 16 hour light and 8 hour darkness cycle with 20 minute dawn and dusk transition periods. The cultures were gently aerated, so as not to disturb the substrate, through narrow bore glass tubes.

The culture vessels were housed in a sealed clear perspex cabinet (breeding box) with cotton sleeves to enable access.

The larvae were fed with Tetramin® flake food at approximately 100 to 500 mg per vessel per day given as a ground powder added to the water surface.
Any gelatinous egg masses produced by breeding adult midges were removed from the culture vessels and transferred to separate vessels and, if required, larvae used to populate new cultures at an initial density of between 100 and 200 larvae.

The diet and diluent were considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study type:

laboratory study

Test type:

not specified

Water media type:

not specified

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Remarks:

The number of emerged adult midges was recorded daily until termination of the test after 28 days.

Hardness:

The water hardness values for the control were determined to be 330 and 350 mg/l as CaCO3 on the Days 0 and 28 respectively, and to range from 184 to 372 and 344 to 364 mg/l as CaCO3 for the test groups on the Days 0 and 28 respectively. The theoretical hardness of the Elendt M4 medium is 250 mg/l as CaCO3. The increase in hardness was considered to be due to the addition of calcium carbonate to the sediment during preparation to adjust the pH to 7.0 ± 0.5.

Test temperature:

Water temperature was maintained at 20ºC to 23ºC throughout the test.

pH:

See Appendix 4 in attached section - Physico-Chemical Measurements

Dissolved oxygen:

See Appendix 4 in attached section - Physico-Chemical Measurements

Salinity:

Not Applicable

Ammonia:

The ammonia concentrations were determined to be 0.443 and 0.060 mg/l as NH4 for the control on the Days 0 and 28 respectively and to range from 0.427 to 0.573 and 0.052 to 1.48 mg/l as NH4 for the test groups on the Days 0 and 28 respectively.

Nominal and measured concentrations:

Based on the results of the range-finding test the following concentrations were assigned to the definitive test: 100, 180, 320, 560 and 1000 mg/kg (dry weight of sediment).

Details on test conditions:

Experimental Preparation
Approximately seven days prior to the start of the test, the test item was prepared by direct addition to the sediment. Homogeneity of mixing trials indicated that extraction of the test item from the prepared ‘wet’ sediment was problematic. Analysis of the prepared ‘dry’ sediment prior to the addition of the water to adjust the moisture content, showed results within 80% to 120% of nominal value. It was considered possible that the addition of water to the prepared sediment may have affected the homogeneity of the prepared sediment. As the prepared ‘dry’ sediment analysis showed near nominal values, the method of preparation was therefore revised in that the prepared ‘dry’ sediment was added directly to the test vessels. In this way it could be assumed that the correct concentration was added to each test vessel. Water was then added to each individual test vessel to adjust the moisture content of the sediment. Analysis could then be performed using the entire sediment in the test vessel without sub-sampling. Analysis of the ‘wet’ sediment prepared in this way also showed low results indicating a possible interaction between the water, calcium carbonate and solvents during extraction.

In order to maintain consistency with the homogeneity trials, and to ensure the correct concentrations were added to each test vessel by sub-sampling the ‘dry’ sediment only, the revised method of preparation was used for the definitive test.

Amounts of test item (100, 180, 320, 560 and 1000 mg) were each separately added to 760 g of quartz sand in a mixing vessel and mixed in a cement mixer for 24 hours after which 200 g of clay and 40 g of air dried peat were added to each vessel and mixed using a cement mixer for a further 24 hours. Each prepared sediment was then further mixed using a Kenwood Chef mixer for approximately 15 minutes to ensure homogeneity. The pH of each prepared sediment was adjusted to 7.0 ± 0.5 with calcium carbonate. The control was prepared in a similar manner, without the addition of test item.

An aliquot (50 g) of the control and each prepared sediment was separately dispensed to 250 ml glass jars. The moisture content of the sediment was adjusted to approximately 40% by adding an aliquot (20 ml) of deionised reverse osmosis water to each jar and carefully mixing to give a 1.5 cm layer of sediment. This was then covered with a 6 cm depth of reconstituted water (sediment:water ratio, 1:4). A plastic disc was placed over the sediment and the reconstituted water poured gently onto the surface of the disc in order to avoid disturbance of the sediment. The disc was removed after addition of the water. The test vessels were then aerated (approximately 1 bubble/second) via narrow bore glass tubes approximately 2 – 3 cm above the sediment layer and the vessels left for seven days prior to addition of the test organisms in order to allow settlement and equilibration of test concentrations between the sediment and water phases.

Four replicates were prepared for each of the control and 100, 180, 320, 560 and 1000 mg/kg test concentrations, plus an additional two replicates of each for sacrificing on Day 10 of the exposure period. A further four replicates were prepared for the control and each test concentration, without the addition of larvae, for sacrificing on Days -7 and 0 for chemical analysis.
Analysis for the concentration of the test item in the ‘dry’ and ‘wet’ sediment was performed on Day -7 (the day of sediment preparation) to confirm correct dosing of the test system (see Appendix 2 - in attached section).
The concentration of the test item in the whole ‘wet’ sediment, interstitial (pore) water and overlying water were verified by chemical analysis on Days 0 and 28 (see Appendix 2 - in attached section).

Analysis was also conducted on the ‘wet’ sediment on Days -7, 0 and 28 after pre-drying the sediment at approximately 60°C (see Appendix 2 - in attached section).

Exposure conditions
In the definitive test 250 ml glass jars were used. After the 7-day equilibration period the aeration was stopped and 20 larvae were placed in each test and control vessel and maintained in a temperature controlled room at 19ºC to 24ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The aeration was switched back on after approximately 24 hours having allowed the larvae to settle in the sediment.

The larvae were fed at a rate of 0.25 to 0.5 Tetramin® flake food per larva per day for the first 10 days and 0.5 to 1.0 mg Tetramin® flake food per larva per day thereafter. The Tetramin® flake food was prepared as a suspension in water and an appropriate volume added to the overlying water.
On Day 10 of the exposure period, two of the extra replicates prepared for the control and each test concentration were sacrificed for the determination of larval survival and weight. The sediment was sieved and live and dead larvae counted. The dry weight of the surviving larvae per test vessel was determined and the mean individual dry weight per vessel calculated.

The measured end-point for the test was the number of live, emerged adult midges. The number of emerged adult midges was recorded daily until termination of the test after 28 days. The sex of the individual midges was also determined after emergence. The male midges were identified by the presence of plumose antennae and the female by the absence of plumose antennae. Any egg masses produced prior to termination were also recorded and removed from the test vessels to prevent re-introduction of larvae into the sediment. The number of visible pupae that failed to emerge were counted separately. Any abnormal behaviour was also recorded.

The control group was maintained under identical conditions but not exposed to the test item.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: Not stated

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

560 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: Not stated

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Remarks on result:

other: Not stated

Details on results:

See any other information on results incl. tables section.

RESULTS

Range-findingTest

Emergence data (number of adult midges emerging from the test vessels) from the exposure of Chironomus riparius larvae to the test item during the range finding test are given in Table 1 - in attached section

Inspection of the data indicated that whilst the 10 and 100 mg/kg test concentrations showed similar emergence to the control, fewer midges emerged in the 1000 mg/kg test concentration compared to the control.

Based on this information test concentrations of 100, 180, 320, 560 and 1000 mg/kg were selected for the definitive test.

DefinitiveTest

Day 10 larval survival and growth

The Day 10 larval survival and growth data during the definitive test are given in Tables 2 and 3 - in attached section

Inspection of the data showed no differences in larval survival between the control and each test group. No significant differences (P≥0.05) were observed in larval growth, in terms of mean larval dry weight, between the control and each test group.

Mortality data

Emergence data (number of adult midges emerging from the test vessels) from the exposure of Chironomus riparius larvae to the test item during the definitive test are given in Table 4 - see in attached section.

Inspection of the emergence data showed similar numbers of emerged adult midges in the control and 100, 180, 320 and 560 mg/kg test concentrations. However, fewer adult midges were observed to have emerged from the 1000 mg/kg test concentration compared to the control. Following normalisation against the control the observed effect at 1000 mg/kg was determined to be less than 50% therefore an EC₅₀value could not be calculated.

Inspection of the emergence data gave the following results based on nominal test concentrations of spiked sediment:

Time (days)	EC50(emergence) mg/kg
28	>1000

The No Observed Effect Concentration after 28 days was 560 mg/kg. The No Observed Effect Concentration was based on no significant reduction in emergence.

There were no observed sub-lethal effects of exposure observed in the definitive test.

The sediment was sieved at the end of the study and the number of visible pupae that failed to emerge were counted (see Table 5 in attached section).

Emergence ratio data

Statistical analysis of the emergence ratio data, transformed by the square root arcsin function in order to obtain an approximate normal distribution and to equalise the variance, using Dunnett’s multiple comparison procedure (Dunnett 1955) (see Appendix 3 in attached section) showed no significant differences (P³0.05) between the control and 100, 180, 320 and 560 mg/kg test groups. However, significant differences were observed between the control and 1000 mg/kg test group.

Development site

The mean development rate per vessel was calculated for each test concentration (see Table 6 in attached section). The overall mean development rates for the control and each test concentration were calculated and an EC₅₀(development rate) estimated by inspection of the data.

The EC₅₀(development) rate based on nominal concentration was estimated to be greater than 1000 mg/kg.

No observed effect concentration

The No Observed Effect Concentration was 560 mg/kg on the basis that no biologically significant reduction in emergence was observed after 28 days and additionally no sub-lethal effects were observed at 560 mg/kg.

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to the sediment-dwelling larvae of Chironomus riparius has been investigated and gave a 28-Day EC50 (emergence) of greater than 1000 mg/kg. The No Observed Effect Concentration was 560 mg/kg. The EC50 (development rate) based on nominal test concentrations was greater than 1000 mg/kg.

Executive summary:

Introduction.

A study was performed to assess the toxicity of the test item ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' to the sediment-dwelling larvae of Chironomus riparius. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 218 (April 2004) “Sediment-Water Chironomid Toxicity Test using Spiked Sediment”.

Methods.

Following a preliminary range-finding test 80 larvae of Chironomus riparius were exposed in groups of twenty (four replicates of 20 larvae per concentration) to formulated sediment spiked with test item over a range of concentrations of 100, 180, 320, 560 and 1000 mg/kg for a period of 28 days. The numbers of emerged adult midges were recorded daily.

A further 40 larvae (two replicates of 20 larvae) of each test group were prepared and sacrificed on Day 10 of the exposure period to determine the 10-Day larval survival and growth data.

Results.

The 28-Day EC₅₀(reduction in emergence) based on nominal test concentrations was greater than 1000 mg/kg. 

The No Observed Effect Concentration was 560 mg/kg.

The EC₅₀(development rate) based on nominal test concentrations was greater than 1000 mg/kg.

Analytical work indicated that extraction of the test item from the prepared ‘wet’ sediment was problematic, possibly due to an interaction between the water, calcium carbonate and solvents during extraction. Analysis was therefore also conducted on the ‘dry’ sediment following mixing with the test item but prior to the addition of the water.

Analysis of the ‘dry’ sediment on the day of preparation (Day -7) showed measured concentrations to range from 92% to 111% of nominal with the exception of the 180 mg/kg test concentration which showed a measured concentration of 73% of nominal value. Analysis of the corresponding duplicate sample stored frozen prior to analysis, showed a measured concentration of 90% of nominal value indicating that the initial analysis was erroneous. The results from the ‘dry’ sediment analysis indicated that the test item had been correctly prepared.

Analysis of the ‘wet’ sediment on Days -7, 0 and 28 showed measured concentrations to range from less than the limit of quantitation to 48% of nominal value. Analysis was also conducted on the ‘wet’ sediment after pre-drying the sediment at approximately 60°C. Analysis of these pre-dried samples on Days -7, 0 and 28 showed measured concentrations to range from 63% to 89% of nominal value.

Analysis of the overlying and interstitial ‘pore’ water on Days 0 and 28 showed measured concentrations of less than the limit of quantitation of the analytical method.

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 16 October 2009 and 25 March 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Six replicates were prepared for the control and 1000 mg/kg test concentration, plus an additional two replicates of each for sacrificing on Day 10 of the exposure period. A further four replicates were prepared for the control and 1000 mg/kg test concentration, without the addition of larvae, for sacrificing on Days -7 and 0 for chemical analysis.

Analysis for the concentration of the test item in the ‘dry’ and ‘wet’ sediment was performed on Day -7 (the day of sediment preparation) to confirm correct dosing of the test system (see attached Appendix 2).

The concentration of the test item in the whole ‘wet’ sediment, interstitial (pore) water and overlying water was verified by chemical analysis on Days 0 and 28 (see attached Appendix 2).

Analysis was also conducted on ‘wet’ sediment on Days -7, 0 and 28 after pre-drying the sediment at approximately 60°C (see attached Appendix 2).

Vehicle:

no

Details on sediment and application:

Approximately seven days prior to the start of the test, the test item was prepared by direct addition to the sediment. Homogeneity of mixing trials conducted under Harlan Laboratories Ltd Project Number: 2289/0058 indicated that extraction of the test item from the prepared ‘wet’ sediment was problematic. Analysis of the prepared ‘dry’ sediment prior to the addition of the water to adjust the moisture content, showed results within 80% to 120% of nominal value. It was considered possible that the addition of water to the prepared sediment may have affected the homogeneity of the prepared sediment. As the prepared ‘dry’ sediment analysis showed near nominal values, the method of preparation was therefore revised in that the prepared ‘dry’ sediment was added directly to the test vessels. In this way it could be considered that the correct concentration was added to each test vessel. Water was then added to each individual test vessel to adjust the moisture content of the sediment. Analysis could then be performed using the entire sediment in the test vessel without sub-sampling. Analysis of the ‘wet’ sediment prepared in this way also showed low results indicating a possible interaction between the water, calcium carbonate and solvents during extraction.
In order to maintain consistency with the homogeneity trials, and to ensure the correct concentrations were added to each test vessel by sub-sampling the ‘dry’ sediment only, the revised method of preparation was used for the definitive test.
An amount of test item (1000 mg) was added to 760 g of quartz sand in a mixing vessel and mixed in a cement mixer for 24 hours after which 200g of clay and 40 g of air dried peat were added to the vessel and mixed using a cement mixer for a further 24 hours. The prepared sediment was then further mixed using a Kenwood Chef mixer for approximately 15 minutes to ensure homogeneity. The pH of the prepared sediment was adjusted to 7.0 ± 0.5 with calcium carbonate. The control was prepared in a similar manner, without the addition of test item.
Aliquots (50 g) of the control and 1000 mg/kg test concentration were separately dispensed to 250 ml glass jars. The moisture content of the sediment was adjusted to approximately 40% by adding an aliquot (20 ml) of deionised reverse osmosis water to each jar and carefully mixing to give a 1.5 cm layer of sediment. This was then covered with a 6 cm depth of reconstituted water (sediment:water ratio, 1:4). A plastic disc was placed over the sediment and the reconstituted water poured gently onto the surface of the disc in order to avoid disturbance of the sediment. The disc was removed after addition of the water. The test vessels were then aerated (approximately 1 bubble/second) via narrow bore glass tubes approximately 2 – 3 cm above the sediment layer and the vessels left for seven days prior to addition of the test organisms in order to allow settlement and equilibration of test concentrations between the sediment and water phases.


Six replicates were prepared for the control and 1000 mg/kg test concentration, plus an additional two replicates of each for sacrificing on Day 10 of the exposure period. A further four replicates were prepared for the control and 1000 mg/kg test concentration, without the addition of larvae, for sacrificing on Days -7 and 0 for chemical analysis.

Test organisms (species):

Chironomus riparius

Details on test organisms:

The sediment-dwelling larvae of Chironomus riparius are representative of a wide variety of natural habitats and can therefore be considered as important non-target organisms in freshwater ecosystems.

The test was carried out using larvae (2 – 3 days old) of Chironomus riparius derived from in-house laboratory cultures.
Larvae were maintained in glass crystalline dishes with a 10 to 20 mm layer of fine quartz sand covered by reconstituted water (Elendt M4) in a temperature controlled room at 21°C to 24°C. The lighting cycle was controlled to give a 16 hour light and 8 hour darkness cycle with 20 minute dawn and dusk transition periods. The cultures were gently aerated, so as not to disturb the substrate, through narrow bore glass tubes. The culture vessels were housed in a sealed clear perspex cabinet (breeding box) with cotton sleeves to enable access.
The larvae were fed with Tetramin® flake food at approximately 100 to 500 mg per vessel per day given as a ground powder added to the water surface.
Any gelatinous egg masses produced by breeding adult midges were removed from the culture vessels and transferred to separate vessels and, if required, larvae used to populate new cultures at an initial density of between 100 and 200 larvae.
The diet and diluent were considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

yes

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

The water hardness values for the control were determined to be 352 and 384 mg/l as CaCO3 on Days 0 and 28 respectively, and 358 and 370 mg/l as CaCO3 for the 1000 mg/kg test group on Days 0 and 28 respectively. The theoretical hardness of the Elendt M4 medium is 250 mg/l as CaCO3. The increase in hardness was considered to be due to the addition of calcium carbonate to the sediment during preparation to adjust the pH to 7.0 ± 0.5.

Test temperature:

Water temperature was maintained at 20 °C to 21 °C throughout the test
Room temperature remained at 19 °C to 24 °C throughout the test and the light intensity was 613 to 788 lux (see attached Appendix 5).

Some of the room temperatures were observed to be slightly in excess of the range given in the study plan of 20 ± 2°C. This deviation was considered not to have affected the outcome or the validity of the test as no adverse effects were observed in the control group throughout the duration of the test.

pH:

7.9 to 8.7 see attached appendix 4 for full details

Dissolved oxygen:

5.1 to 9.3 mg O2/l see attached appendix 4 for full details

Salinity:

Not applicable

Ammonia:

The ammonia concentrations were determined to be 0.354 and 0.034 mg/l as NH4 for the control on Days 0 and 28 respectively and 0.100 and 0.034 mg/l as NH4 for the 1000 mg/kg test group on Days 0 and 28 respectively.

Nominal and measured concentrations:

In the range-finding test Chironomus riparius larvae were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/kg (dry weight of sediment)
Based on the results of the range-finding test a "limit test" was conducted for the definitive test at a concentration of 1000 mg/kg (dry weight of sediment) to confirm that at the maximum test concentration given in the OECD/EC test guidelines no mortalities or sub-lethal effects of exposure were observed.

Details on test conditions:

Test Water
The reconstituted water (Elendt M4) used for the range-finding and definitive tests was the same as that used to maintain the stock animals. The reconstituted water is defined in the attached Appendix 1.

Formulated Sediment
A defined formulated sediment was used with the following composition:
Industrial quartz sand 76% w/w
Kaolinite clay 20% w/w
Sphagnum moss peat 4% w/w
The peat was air dried and homogenised to give a particle size of less than 1 mm. The organic carbon content of the final mixture was 1.9%.


Range-finding test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test Chironomus riparius larvae were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/kg (dry weight of sediment). Approximately seven days prior to the start of the range-finding test the test item was incorporated directly into the sediment.
Amounts of test item (5.0, 50 and 500 mg) were each separately added to 380 g of quartz sand in a mixing vessel and mixed in a cement mixer for 24 hours after which 100 g of clay and 20 g of air dried peat were added to each vessel and mixed using a cement mixer for a further 24 hours. An aliquot (196 ml) of deionised reverse osmosis water was added to each sediment and mixed using a Kenwood Chef mixer to give the 10, 100 and 1000 mg/kg test concentrations respectively with nominal moisture content of approximately 40% of dry weight. The pH of each prepared sediment was adjusted to 7.0 ± 0.5 with calcium carbonate. The control was prepared in a similar manner, without the addition of test item.
Each prepared sediment was dispensed to glass beakers (600 ml) to give a 2 cm layer and was then covered with an 8 cm depth of reconstituted water (sediment:water ratio, 1:4). A plastic disc was placed over the sediment and the reconstituted water poured gently onto the surface of the disc in order to avoid disturbance of the sediment. The disc was removed after addition of the water. Two replicates were prepared for the control and each test concentration. The test vessels were then aerated (approximately 1 bubble/second) via narrow bore glass tubes approximately 2 to 3 cm above the sediment layer and the vessels left for approximately seven days prior to addition of the test organisms in order to allow settlement and equilibration of test concentrations between the sediment and water phases.
After the 7-Day equilibration period the aeration was stopped and 20 larvae were placed in each test and control vessel. The vessels were maintained in a temperature controlled room at 21°C to 23°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. Some of the room temperatures were observed to be slightly in excess of the range given in the study plan of 20 ± 2°C. This deviation was considered not to have affected the outcome or the validity of the test as no adverse effects were observed in the control group throughout the test.
The aeration was switched back on after approximately 24 hours having allowed the larvae to settle in the sediment.
The larvae were fed at a rate of 0.25 to 0.5 mg Tetramin® flake food per larva per day for the first 10 days and 0.5 to 1.0 mg Tetramin® flake food per larva per day thereafter. The Tetramin® flake food was prepared as a suspension and an appropriate volume added to the overlying water.
The measured end-point for the test was the number of live, emerged adult midges. The number of emerged adult midges was recorded daily until termination of the test after 28 days. The sex of the individual midges was also determined after emergence. The male midges were identified by the presence of plumose antennae and the female by the absence of plumose antennae.
The control group was maintained under identical conditions but not exposed to the test item.

Exposure conditions
After the 7-Day equilibration period the aeration was stopped and 20 larvae were placed in each test and control vessel and maintained in a temperature controlled room at 19°C to 24°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The aeration was switched back on after approximately 24 hours having allowed the larvae to settle in the sediment.
The larvae were fed at a rate of 0.25 to 0.50 mg Tetramin® flake food per larva per day for the first 10 days and 0.50 to 1.0 mg Tetramin® flake food per larva per day thereafter. The Tetramin® flake food was prepared as a suspension in water and an appropriate volume added to the overlying water.
On Day 10 of the exposure period, two of the extra replicates prepared for the control and 1000 mg/kg test concentration were sacrificed for the determination of larval survival and weight. The sediment was sieved and live and dead larvae counted. The dry weight of the surviving larvae per test vessel was determined by drying in an oven at approximately 105°C and the mean individual dry weight per vessel calculated.
The measured end-point for the test was the number of live, emerged adult midges. The number of emerged adult midges was recorded daily until termination of the test after 28 days with the exception of Day 5 for the 1000 mg/kg test concentration when the numbers were not recorded in error. This was considered not to affect the integrity of the test given that midge emergence would not be expected at this stage of the test. The sex of the individual midges was also determined after emergence. The male midges were identified by the presence of plumose antennae and the females by the absence of plumose antennae. Any egg masses produced prior to termination were also recorded and removed from the test vessels to prevent re-introduction of larvae into the sediment. The number of visible pupae that failed to emerge were counted separately. Any abnormal behaviour was also recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Physico-chemical measurements
Room temperature and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations, water temperature and pH were recorded daily in each test vessel throughout the test. The pH and the dissolved oxygen concentration were measured using a Hach HQ30d pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The water hardness and ammonia concentration were determined in one vessel from the control and 1000 mg/kg test concentration on Days 0 and 28.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: 95 % CL not reported

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: 95 5 CL not reported

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Remarks on result:

other: 95 % CL not reported

Details on results:

Range-finding Test
Emergence data (number of adult midges emerging from the test vessels) from the exposure of Chironomus riparius larvae to the test item during the range finding test are given in the attached Table 1.
There was no observed reduction in emergence of adult midges observed at any test concentrations employed in the range-finding test.
Based on this information, a single test concentration of six replicates, at 1000 mg/kg was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD test guidelines no mortalities or sub-lethal effects of exposure were observed.

Definitive Test
Day 10 larval survival and growth
The Day 10 larval survival and growth data during the definitive test are given in the attached Tables 2 and 3.
Inspection of the data showed no significant differences in larval survival and growth, in terms of mean larval dry weight, between the control and 1000 mg/kg test group.
Emergence data
Emergence data (number of adult midges emerging from the test vessels) from the exposure of Chironomus riparius larvae to the test item during the definitive test are given in the attached Table 4.
There was no significant reduction in emergence of adult midges observed at the 1000 mg/kg test concentration.
Inspection of the emergence data gave the following results based on nominal test concentrations of spiked sediment:
Time (days) EC50 (emergence) mg/kg
28 >1000

The No Observed Effect Concentration after 28 days was 1000 mg/kg. The No Observed Effect Concentration was based on no significant reduction in emergence.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/kg.
There were no observed sub-lethal effects of exposure observed in the definitive test.
The sediment was sieved at the end of the test and the number of visible pupae that failed to emerge were counted (see attached Table 5). No larvae were observed.

Emergence ratio data
Statistical analysis of the emergence ratio data, transformed by the square root arcsin function in order to obtain an approximate normal distribution and to equalise the variance, using a Students t-test (see attached Appendix 3) showed no significant difference (P≥0.05) between the control and 1000 mg/kg test group.
Development rate
The mean development rate per vessel was calculated for each test concentration (see attached Table 6). The overall mean development rates for the control and 1000 mg/kg test concentration were calculated and an EC50 (development rate) estimated by inspection of the data.
Inspection of the development rate data gave the following results based on nominal test concentrations of spiked sediment:
Time (days) EC50 (emergence) mg/kg
28 >1000

No observed effect concentration
The No Observed Effect Concentration was 1000 mg/kg on the basis that no biologically significant reduction in emergence was observed after 28 days and additionally no sub-lethal effects were observed at 1000 mg/kg.

Physico-chemical measurements
The results of the physico-chemical measurements are given in the attached Appendix 4. Water temperature was maintained at 20°C to 21°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Room temperature remained at 19°C to 24°C throughout the test and the light intensity was 613 to 788 lux (see attached Appendix 5).
Some of the room temperatures were observed to be slightly in excess of the range given in the study plan of 20 ± 2°C. This deviation was considered not to have affected the outcome or the validity of the test as no adverse effects were observed in the control group throughout the duration of the test.
The water hardness values for the control were determined to be 352 and 384 mg/l as CaCO3 on Days 0 and 28 respectively, and 358 and 370 mg/l as CaCO3 for the 1000 mg/kg test group on Days 0 and 28 respectively. The theoretical hardness of the Elendt M4 medium is 250 mg/l as CaCO3. The increase in hardness was considered to be due to the addition of calcium carbonate to the sediment during preparation to adjust the pH to 7.0 ± 0.5.
The ammonia concentrations were determined to be 0.354 and 0.034 mg/l as NH4 for the control on Days 0 and 28 respectively and 0.100 and 0.034 mg/l as NH4 for the 1000 mg/kg test group on Days 0 and 28 respectively.

Verification of test concentrations
Analytical work conducted under Harlan Laboratories Ltd Project Number: 2289/0058 indicated that extraction of the test item from the prepared ‘wet’ sediment was problematic, possibly due to an interaction between the water, calcium carbonate and solvents during extraction. Analysis was therefore also conducted on the ‘dry’ sediment following mixing with the test item but prior to the addition of the water.
Analysis of the ‘dry’ sediment (see attached Appendix 2) on the day of preparation (Day -7) showed a measured concentration of 93% of nominal value indicating that the test item had been correctly prepared.
Analysis of the ‘wet’ sediment (see attached Appendix 2) on Days -7, 0 and 28 showed measured concentrations to range from 51% to 57% of nominal value. Analysis was also conducted on the ‘wet’ sediment after pre-drying the sediment at approximately 60°C. Analysis of these pre-dried samples on Days -7, 0 and 28 showed measured concentrations to range from 50% to 54% of nominal value.
Analysis of the overlying and interstitial ‘pore’ water (see attached Appendix 2) on Days 0 and 28 showed measured concentrations of less than the limit of quantitation of the analytical method.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Data Evaluation
An estimate of the 28-Day EC50 (reduction in emergence) value was given by inspection of the data.
Statistical analysis of the emergence data was performed using a Students t-test. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001). Analysis was performed on the emergence ratio (ER) values. The emergence ratio was calculated for each control and test vessel as described in the attached “Data Evaluation”

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to the sediment-dwelling larvae of Chironomus riparius has been investigated and gave a 28-Day EC50 (emergence) of greater than 1000 mg/kg. The No Observed Effect Concentration was 1000 mg/kg. The EC50 (development rate) based on nominal test concentrations was greater than 1000 mg/kg.

Executive summary:

Introduction.

A study was performed to assess the toxicity of the test item to the sediment-dwelling larvae of Chironomus riparius. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 218 (April 2004) “Sediment-Water Chironomid Toxicity Test using Spiked Sediment”.

Methods.

Following a preliminary range-finding test, 120 larvae of Chironomus riparius (six replicates of 20 larvae) were exposed to formulated sediment spiked with test item at a single concentration of 1000 mg/kg (dry weight of sediment) for a period of 28 days. The numbers of emerged adult midges were recorded daily.

A further 40 larvae (two replicates of 20 larvae) of the control and 1000 mg/kg test group were prepared and sacrificed on Day 10 of the exposure period to determine the 10-Day larval survival and growth data.

Results.

The 28-Day EC₅₀(reduction in emergence) based on nominal test concentrations was greater than 1000 mg/kg. The No Observed Effect Concentration was 1000 mg/kg.

The EC₅₀(development rate) based on nominal test concentrations was greater than 1000 mg/kg.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/kg.

Analytical work conducted under Harlan Laboratories Ltd Project Number: 2289/0058 indicated that extraction of the test item from the prepared ‘wet’ sediment was problematic, possibly due to an interaction between the water, calcium carbonate and solvents during extraction. Analysis was therefore also conducted on the ‘dry’ sediment following mixing with the test item but prior to the addition of the water.

Analysis of the ‘dry’ sediment on the day of preparation (Day -7) showed a measured concentration of 93% of nominal value indicating that the test item had been correctly prepared.

Analysis of the ‘wet’ sediment on Days -7, 0 and 28 showed measured concentrations to range from 51% to 57% of nominal value. Analysis was also conducted on the ‘wet’ sediment after pre-drying the sediment at approximately 60°C. Analysis of these pre-dried samples on Days -7, 0 and 28 showed measured concentrations to range from 50% to 54% of nominal value.

Analysis of the overlying and interstitial ‘pore’ water on Days 0 and 28 showed measured concentrations of less than the limit of quantitation of the analytical method.

Reference 3

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 15 October 2009 and 8 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals (October 2007) No 225 "Sediment-water Lumbriculus Toxicity Test using Spiked Sediment"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Verification of test concentrations
Samples of the freshly prepared sediments were taken from the control and each test concentration on Day-9 for quantitative analysis.
Samples of the overlying water and sediment were taken from the control and each test concentration on Day 0 for quantitative analysis. Samples of the overlying water and sediment were taken from the control and the 10 and 1000 mg/kg test concentrations on Day 28 for quantitative analysis

For the sediment analysis two samples of the control and the appropriate test concentration were taken at each occasion. One sample was analysed ‘wet’ as sampled. The other sample was dried at approximately 60oC for approximately 24 hours prior to analysis.
Analysis for the concentration of test item in the interstitial water of the control and each appropriate test concentration was also performed on Days 0 and 28. The interstitial water was isolated by centrifugation (10000 g at 4°C for 30 minutes).
Duplicate samples, where possible, were taken and stored at approximately -20ºC for further analysis if necessary.

Vehicle:

yes

Details on sediment and application:

Test Water
The test water used for the definitive test was the same as that used to maintain the new stock cultures. Laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was 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.

Formulated Sediment
A defined formulated sediment was used with the following composition:
Industrial quartz sand 75% w/w
Kaolinite clay 20% w/w
Spagnum peat, ground and air dried 4.5% w/w
Food source 0.5% w/w
The organic carbon content of the final mixture was 2.1%.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

Test Species
The test was carried out using adult worms of Lumbriculus variegatus derived from in-house cultures.
The worms were maintained in 40 litre glass aquaria with a substrate of shredded paper hand towels. Dechlorinated tap water was continuously passed through the tank. The culture vessels were maintained in a temperature controlled room at approximately 21ºC. The lighting cycle was controlled to give a 16 hour light and 8 hour darkness cycle with 20 minute dawn and dusk transition periods. The cultures were gently aerated so as not to disturb the substrate, through narrow bone glass tubes.
The worms were fed with Tetramin® flake food at approximately 30 ml of a 5 g/100 ml dispersion every day. The Tetramin® flake food was prepared as a suspension in water and an appropriate volume added to the overlying water.
The diet and diluent were considered not to contain any contamination that would effect the integrity or outcome of the test.
Approximately 10 -14 days prior to exposure of the test organisms, sufficient numbers of worms were dissected in the median body region with a scalpel. The posterior ends were placed in culture medium with a small amount of culturing substrate and left to regenerate new heads. This was conducted to synchronise the age of the worms.
A positive control (Harlan Laboratories Ltd., Project Number: 41102162) used pentachlorophenol sodium salt (PCP-Na salt) as the reference item. Details of the positive control are given in Appendix 2. The positive control was conducted between 22 June 2011 and 1 August 2011.

Study type:

laboratory study

Test type:

other: A PROLONGED TOXICITY TEST USING SPIKED SEDIMENT WITH THE OLIGOCHAETE, Lumbriculus variegatus

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

The water hardness and conductivity were required to be determined in one replicate vessel for the control and highest test concentration at the start and end of the exposure period. Due to a technical error, no results were available for the hardness on Day 0 and the conductivity was not recorded on Day 28. This was considered not to affect the integrity of the study as no adverse effects were observed in the control. The water hardness was measured using the methods described in Guide to Field and On-site Test Methods for the Analysis of Waters (British Standards Institution 1993). The conductivity was measured using a Hach HQ30d Flexi Handheld meter.

Test temperature:

The water temperature was also recorded in one control vessel (replicate R1) every hour using a Testo temperature logger.

pH:

The pH of the sediment during preparation was measured using a IQ 150 pH meter

Dissolved oxygen:

The pH and oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter and the temperature measured using a Hanna Instruments HI 93510 digital thermometer.

Salinity:

Not applicable

Ammonia:

Total ammonia content was measured in one replicate vessel for the control and each test concentration at the start of the test and three times a week thereafter.

Details on test conditions:

Definitive test
Initial tests were conducted using a method of preparation whereby the test item was initially mixed with the sand component of the artificial sediment and then mixed with the remaining components. Once mixed, the sediments were placed in the test vessels, overlying water added and left for 2 days to equilibrate. Numerous attempts were made to complete the test but in each instance the worm survival was poor and failed to meet the validation criteria. An extensive investigation indicated that it may be increased ammonia levels in the test vessels during the test which may have contributed to the poor worm survival rate. Consequently, the method of preparation was revised to incorporate pre-conditioning of the peat prior to use. Investigations were conducted using the revised method of preparation and monitoring ammonia levels without the addition of test organisms. This indicated that ammonia levels remained low. Positive control studies were run using the revised sediment preparation method which showed good increases in worm numbers and met the validation criteria. Due to the problems associated with homogenous mixing of test items of the type used for this study, in agreement with the Sponsor, the preparation method was further revised as described in Section "Experimental preparation"

Experimental preparation
For the purpose of the definitive test, the test item was incorporated directly into the sediment.
Prior to dosing, the air dried peat component of the artificial sediment was pre-conditioned. An amount of air dried peat (67.5 g) was added to 600 ml of deionised reverse osmosis water, the pH adjusted to approximately 5.5 and then stirred using a magnetic stirrer for 2 days. After 2 days pre-conditioning, the pH was again adjusted to approximately 6 ± 0.5. Six lots were prepared, one for the control and each test concentration.
Approximately 24 hours prior to the end of the pre-conditioning period of the peat, the test item was mixed with the sand component of the artificial sediment. Amounts of test item (15, 48, 150, 480 and 1500 mg) were each separately added to 1125 g of quartz sand and mixed for approximately 24 hours using a cement mixer.
After approximately 24 hours mixing, each sand/test item mixture was separately mixed using a Kenwood Chef mixer with the 67.5 g pre-conditioned peat suspension and 300 g of kaoline clay. Each prepared sediment was then placed in a 3 litre beaker, filled with dechlorinated tap water at a ratio of 1:4 (sediment:water), aerated at a rate of approximately 3 bubbles per second and left to pre-condition for approximately 7 days.
Following the second pre-conditioning period, the overlying water was removed from each prepared sediment and the food source (3.75 g each of dried nettles and α-cellulose) using a Kenwood Chef mixer to give the final test concentrations of 10, 32, 100, 320 and 1000 mg/kg with a nominal moisture content of 40% dry weight.

Each prepared sediment (approximately 2 cm) was then dispersed to 300 ml glass beakers. An 8 cm layer of dechlorinated tap water was then added to each vessel. A plastic disc was placed over the sediment and water poured gently onto the disc in order to avoid disturbance of the sediment. The plastic disc was then removed. The test vessels were then left on aeration (approximately 1 bubble per second via narrow bore glass tubes) for 2 days prior to addition of the test organisms to allow settlement and equilibration of test concentrations between the sediment and water phases.
The control was prepared in an identical manner without the addition of test item.
Six replicate test vessels were prepared for the control and 1000 mg/kg test concentration along with four replicate test vessels for the 10, 32, 100 and 320 mg/kg test concentrations. An additional four replicates were prepared for the control, 10 and 1000 mg/kg test concentrations for sacrificing on Days 0 and 28 for chemical analysis. An additional two replicates were prepared for the 32, 100 and 320 mg/kg test concentrations for sacrificing on Day 0 for chemical analysis.
The concentration of the test item in the test sediment was determined on Days -9 (the day the sediment was prepared), 0 and 28 and from the overlying water and interstitial water on Days 0 and 28 (see Appendix 3).

Exposure conditions
In the definitive test 300 glass beakers were used.  After the two day equilibration period 10 worms were placed in each test and control vessel (replicates R1 – R6 for the control and 1000 mg/kg test concentration and replicates R1 – R4 for the 10, 32, 100 and 320 mg/kg test concentrations) and maintained in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk periods.
On Day 0 a representative 10 worms were sampled and dried at approximately 60°C for approximately 24 hours to determine the dry weight.  This was a deviation to the study plan which stated that the worms should be dried at 100oC ± 5oC for approximately 24 hours.  This was considered not to affect the integrity of the test as the drying temperature and period used was considered sufficient to completely dry the worms.  This showed an average dry weight per worm of 0.22 mg.
Losses of overlying water due to evaporation were adjusted daily, if necessary, by the addition of deionised water.
The measured end-point for the test was the number of live worms.  The number of live worms in each replicate was determined on Day 28.  For each test vessel, a sediment slurry was formed, poured into a shallow tray and the worms removed by tweezers and counted.
The dry weight of surviving worms from each replicate was determined at the end of the test. The worms were dried at approximately 60 oC for approximately 72 hours. This was a deviation to the study plan which stated that the worms should be dried at 100 oC ± 5 oC for approximately 24 hours.  This was considered not to affect the integrity of the test as the drying temperature and period used was considered sufficient to completely dry the worms.
The control group was maintained under identical conditions but not exposed to the test item.
Observations on the general condition of the worms in each vessel were made daily

Reference substance (positive control):

yes

Remarks:

pentachlorophenol sodium salt (PCP-Na salt)

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

590 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% 310 to greater than 1000mg/kg

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% not stated

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

320 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% not stated

Details on results:

Definitive Test
Survival and reproduction data
The number of surviving worms and dry weight data recorded on Day 28 from the exposure of Lumbriculus variegatus to the test item are given in Tables 1 and 2 respectively.
Analysis of the numbers of surviving worms at the end of the test by the Maximum Likelihood-Probit method (Finney 1971) based on nominal test concentrations gave the following results.
Time (days) EC50 (mg/kg) 95% Confidence limits
(mg/kg)
28 590 310 - >1000

Statistical analysis (see Appendix 4) of the numbers of surviving worms at the end of the test was carried out for the control and each test group using one way analysis of variance incorporating Bartlett’s Test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no significant differences (P≥0.05) between the control and the 10, 32 and 100 mg/kg test groups in terms of the number of surviving worms at the end of the test. However, the 320 and 1000 mg/kg test groups were significantly different (P<0.05) from the control in terms of the number of surviving worms at the end of the test. Therefore the No Observed Effect Concentration (NOEC) was 100 mg/kg (dry weight of sediment).
The Lowest Observed Effect Concentration (LOEC) was considered to be 320 mg/kg (dry weight of sediment).
Statistical analysis of the worm weight data at the end of the test was carried out for the control and each test group using one way analysis of variance incorporating Bartlett’s Test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) (see Appendix 4). There were no significant differences (P0.05) between the control, 10, 32, 100, 320 and 1000 mg/kg test groups despite significant differences in worm numbers at 320 and 1000 mg/kg indicating that growth may not be affected.

Physico-chemical measurements
The pH values of the sediments prepared on Day -9 of the test are given in Appendix 5.
The results of the physico-chemical measurements of the overlying water are given in Appendix 6. Water temperature was maintained at approximately 21ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH. Water temperature was also monitored in control replicate R1 throughout the test and showed to water temperature to be 20 ± 2oC (see Figure 1).
Room temperature remained at 20ºC to 23ºC throughout the test and the light intensity was 396 to 470 lux (see Appendix 7).
The water hardness for the control and 1000 mg/kg test concentration was determined to be 262 and 278 mg/l as CaCO3 respectively on Day 28.
The conductivity for the control and 1000 mg/kg test concentration was determined to be 543 and 526 µS/cm respectively on Day 0.
The ammonia concentrations were determined to range from 0.050 to 4.06 mg/l as NH4 for the control and from 0.010 to 3.85 mg/l as NH4 for the test concentrations (see Appendix 8). The increase in ammonia during the test period was considered to be due to waste products produced by the worms as a result of feeding.

Observations on the test organisms
No abnormalities or sub-lethal effects of exposure to the test item were observed throughout the duration of the test.

Observations on the test sediment and Overlying Water
No abnormalities were observed in the sediment layer or overlying water.

Verification of test concentrations
Analysis of the 100, 320 and 1000 mg/kg ‘wet’ sediment (see Appendix 3) on Days -9 (i.e. when the sediment was prepared and prior to the pre-conditioning period), 0 and 28 showed measured concentrations to range from 18% to 52% of nominal. These low values were due to difficulties extracting the test item from the wet sediment. The solvent extractions from the wet sediment were hindered by the addition of calcium carbonate which was used to neutralise the humic acid component of the sediment. When wetted, this became an impenetrable paste to hydrocarbon solvent extraction and so resulted in poor recoveries.
Samples of the sediment were taken on Days -9, 0 and 28 and dried at approximately 60oC for approximately 24 hours prior to analysis. Analysis of the 100, 320 and 1000 mg/kg ‘dry’ sediments on Day -9 and 0 showed measured concentrations to range from 68% to 78% of nominal and from 28% to 38% of nominal respectively. Analysis of the 1000 mg/kg ‘dry’ sediment on Day 28 showed a measured concentration of 51% of nominal. The results were below the 80% acceptance level which was considered to be due to loss due to the volatile nature of the test item. Analysis of the 10 and 32 mg/kg test concentrations showed variable results. This was considered to be due these concentrations being below the limit of quantitation of the analytical method which was assessed as 2.9 mg/kg.
Analysis of the interstitial (pore) water on Day 0 showed measured concentrations to be less than the limit of quantitation (LOQ) of the analytical method (assessed as 4.9 mg/l). Analysis of the interstitial (pore) water from the 10 mg/kg test concentration on Day 28 showed the measured concentration to be less than the LOQ. Analysis of the interstitial (pore) water from the 1000 mg/kg test concentration on Day 28 showed a measured concentration of 7.05 mg/l.
Analysis of the overlying water on Days 0 and 28 showed measured concentrations to be less than the (LOQ) of the analytical method (assessed as 0.49 mg/l).

Results with reference substance (positive control):

A positive control used pentachlorophenol sodium salt (PCP-Na salt) as the reference item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/kg (dry weight of sediment). (Please see in any other information materials and method section for positive control).

Reported statistics and error estimates:

Evaluation of data
The 28-Day EC50 (reproduction) value and associated confidence limits were calculated by the Maximum Likelihood-Probit method (Finney 1971) using ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
For the estimation of the “Lowest Observed Effect Concentration” (LOEC) and the “No Observed Effect Concentration” (NOEC) the numbers of surviving worms (data transformed by log10 function to ensure homogeneity of variance) and the dry worm weight data (data transformed by log10 function to ensure homogeneity of variance) at the end of the test for the control and each test group were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) (see Appendix 4). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table1              Number of SurvivingWormsin the Definitive Testafter 28 Days

Nominal Concentration (mg/kg)	Number of Surviving
	Day 28
	R1	R2	R3	R4	R5	R6
Control	43	36	28	30	33	36
10	36	36	33	40	-	-
32	31	36	33	39	-	-
100	34	32	32	27	-	-
320	24	17	18	23	-	-
1000	15	11	11	15	11	13

   - Replicates not prepared

Table2              Worm Weight in the Definitive Test after 28 Days

Nominal Concentration (mg/kg)	Mean Individual Worm Dry Weight (mg)
	Replicate
	1	2	3	4	5	6
Control	1.23	1.38	1.66	1.31	1.37	1.36
10	1.19	1.28	1.56	1.54	-	-
32	0.53	1.51	1.59	1.89	-	-
100	1.29	1.38	1.42	1.74	-	-
320	1.33	2.05	2.01	1.51	-	-
1000	1.55	1.37	1.34	1.31	1.30	0.92

R₁– R₆= Replicates 1 to 6

- Replicates not prepared

 

Appendix 4 Statistical Analysis

Analysis of Numbers of Surviving

Analysis of the number of surviving worms obtained at termination of the test from the control and each test group were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955).

The number of surviving worms of the control and each test group were used for this analysis. The analysis was performed using the individual number of surviving worms per replicate for each test group. The mean and standard deviations calculated from this data are given in the table below. The data was transformed by log₁₀function to ensure homogeneity of variance prior to statistical analysis.

Nominal Concentration (mg/kg)	Mean Individual Worm Weight (mg)
Control Rl-R6	Mean                                           1.39 Standard Deviation                       0.15
10 Rl-R4	Mean                                            1.39 Standard Deviation                        0.18
32 Rl- R4	Mean                                            1.38 Standard Deviation                       0.59
100 Rl- R4	Mean                                            1.46 Standard Deviation                        0.19
320 Rl- R4	Mean                                             1.73 Standard Deviation                         0.36
1000 Rl- R6	Mean                                            1.30 Standard Deviation                        0.21

No significant differences (P³0.05) were found between the control, 10, 32 and 100 mg/kg test groups using the above methods of statistical analysis. However, significant differences (P<0.05) were found between the control and the 320 and 1000 mg/kg test groups.

Appendix 4 (continued) Statistical Analysis

Analysis of Worm Dry Weight Data

Analysis of the worm dry weight data obtained at termination of the test from the control and each test group were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955).

The worm weight data of the control and each test group were used for this analysis. The analysis was performed using the individual mean worm weight values, from each replicate for each test group. The mean and standard deviations calculated from this data are given in the table below. The data was transformed by log₁₀function to ensure homogeneity of variance prior to statistical analysis.

Nominal Concentration (mg/kg)	Mean Individual Worm Weight (mg)
ControlR1– R6	Mean	1.39
	Standard Deviation	0.15
10 R1– R4	Mean	1.39
	Standard Deviation	0.18
32 R1– R4	Mean	1.38
	Standard Deviation	0.59
100 R1– R4	Mean	1.46
	Standard Deviation	0.19
320 R1– R4	Mean	1.73
	Standard Deviation	0.36
1000 R1– R6	Mean	1.30
	Standard Deviation	0.21

No significant differences (P³0.05) were found between the control and each test group using the above methods of statistical analysis. 

 

R₁– R₆= Replicates 1 to 6

*Significant differences

R₁– R₆= Replicates 1 to 6

Appendix 5      pH Values of Prepared Test Sediments

Nominal Concentration (mg/kg)	pH Values of Prepared Test Sediment
Control	6.03
10	6.24
32	6.50
100	6.74
320	6.87
1000	6.94

 

Appendix 6      Physico-Chemical Measurements

Nominal Concentration (mg/kg)		Day 0					Day 7					Day 14					Day 21					Day 28
		pH		mg O2/l		%ASV*		T°C		pH		mg O2/l		% ASV*		T°C		pH		mg O2/l		% ASV*		T°C		pH		mg O2/l		% ASV*		TºC		pH		mg O2/l		% ASV*		TºC
Control		8.1		6.4		74		22		8.5		8.5		98		22		8.3		8.4		97		22		8.7		8.4		97		22		8.3		8.5		96		21
10		8.1		6.2		71		22		8.6		8.5		98		22		8.6		8.4		97		22		8.7		8.3		95		22		8.4		8.7		96		20
32		8.1		6.0		69		22		8.4		8.4		97		22		8.6		8.4		97		22		8.7		8.2		94		22		8.5		8.6		95		20
100		8.1		6.2		71		22		8.4		8.0		92		22		8.5		8.3		95		22		8.7		8.2		94		22		8.5		8.6		95		20
320		8.0		6.5		75		22		8.5		8.2		94		22		8.6		8.3		95		22		8.6		8.0		92		22		8.5		8.6		97		21
1000		8.1		6.4		74		22		8.6		8.3		95		22		8.7		8.4		97		22		8.7		8.1		93		22		8.6		8.6		97		21

 

Appendix7      Room Temperature and Light Intensity Records

Day	Room Temperature (°C)		Light Intensity (Lux)
	Maximum	Minimum
0	22	22	436
1	22	21	427
2	22	21	430
3	22	21	413
4	22	21	411
5	22	22	398
6	22	22	402
7	22	22	403
8	22	22	410
9	22	22	428
10	22	22	396
11	22	21	397
12	22	21	406
13	22	22	401
14	22	22	399
15	22	21	419
16	22	21	406
17	22	21	411
18	22	22	404
19	22	20	407
20	22	21	457
21	23	21	399
22	23	21	415
23	23	21	408
24	22	20	470
25	23	21	423
26	22	21	417
27	22	21	460
28	22	20	429

 

Appendix 8      Ammonia Determination

Day	Ammonia Concentration (NH4) mg/l
	Control	10 mg/kg	32 mg/kg	100 mg/kg	320 mg/kg	1000 mg/kg
0	0.206	0.087	0.078	0.168	0.097	0.138
3	1.59	1.16	1.05	1.71	0.904	1.06
5	2.96	1.59	1.68	3.27	0.141	1.20
7	4.06	2.61	2.32	3.85	0.192	2.17
10	3.82	2.52	1.97	3.48	1.19	3.28
12	3.54	1.91	0.690	2.84	1.42	1.51
14	1.12	0.751	0.234	2.39	0.529	0.411
17	0.137	0.265	0.425	0.266	0.521	0.035
19	0.122	0.046	0.221	0.087	0.267	0.049
21	0.050	0.078	0.010	0.014	0.078	0.096
24	0.561	0.193	0.546	0.049	0.100	0.012
26	0.488	0.042	0.054	0.022	0.014	0.014
28	0.094	0.057	0.061	0.048	0.052	0.030

*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

Measurements taken from Replicate 1 from each test group

Validity criteria fulfilled:

yes

Remarks:

The test was designed to evaluate toxicity of single substances, not complex mixtures such as the test item which is considered as an UVCB substance. This means that greater care should be taken in the interpretation of the data.

Conclusions:

The Day 28 EC50 based on nominal test concentrations was was 590 mg/kg with 95% confidence limits of 310 to greater than 1000 mg/kg. The No Observed Effect Concentration was 100 mg/kg and the Lowest Observed Effect Concentration was 320 mg/kg.

Executive summary:

Introduction

A study was performed to assess the toxicity of the test item to the sediment-dwelling oligochaete, Lumbriculus variegatus. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (October 2007) No 225 "Sediment-water Lumbriculus Toxicity Test using Spiked Sediment".

Methods

During the test, 40 to 60 worms of Lumbriculus variegatus (4 to 6 replicates of 10 worms) were exposed to formulated sediment spiked with test item at concentrations of 10, 32, 100, 320 and 1000 mg/kg (dry weight of sediment) for a period of 28 days. The numbers of worms and the dry weight data of these worms were recorded at the end of the test.

Further replicates were prepared for the control and each test group and sacrificed on Days 0 and 28 for chemical analysis of the sediment and overlying water.

Results

The Day 28 EC₅₀based on nominal test concentrations was 590 mg/kg with 95% confidence limits of 310 to greater than 1000 mg/kg. The No Observed Effect Concentration was 100 mg/kg and the Lowest Observed Effect Concentration was 320 mg/kg.

Analysis of the 100, 320 and 1000 mg/kg ‘wet’ sediment on Days -9 (i.e. when the sediment was prepared and prior to the pre-conditioning period), 0 and 28 showed measured concentrations to range from 18% to 52% of nominal. These low values were due to difficulties extracting the test item from the wet sediment. The solvent extractions from the wet sediment were hindered by the addition of calcium carbonate which was used to neutralise the humic acid component of the sediment.  When wetted, this became an impenetrable paste to hydrocarbon solvent extraction and so resulted in poor recoveries.

Samples of the sediment were taken on Days -9, 0 and 28 and dried at approximately 60^oC for approximately 24 hours prior to analysis. Analysis of the 100, 320 and 1000 mg/kg ‘dry’ sediments on Day -9 and 0 showed measured concentrations to range from 68% to 78% of nominal and from 28% to 38% of nominal respectively. Analysis of the 1000 mg/kg ‘dry’ sediment on Day 28 showed a measured concentration of 51% of nominal. The low results could be attributed to a number of factors including the problems encountered with the analytical method, binding of the test item to the sediment matrix, the volatile nature of the test item and the problems associated with the mixing of this type of test item with the sediment. A study conducted to determine the degradation rate of the test item in soil showed a DT₅₀of 22.4 days in an aerobic soil system,therefore losses of test item over the duration of the test may also have been due, in part, to degradation of the test item in the test system. Analysis of the 10 and 32 mg/kg test concentrations showed variable results. This was considered to be due these concentrations being below the limit of quantitation of the analytical method which was assessed as 2.9 mg/kg.

Analysis of the interstitial (pore) water on Day 0 showed measured concentrations to be less than the limit of quantitation (LOQ) of the analytical method (assessed as 4.9 mg/l). Analysis of the interstitial (pore) water from the 10 mg/kg test concentration on Day 28 showed the measured concentration to be less than the LOQ. Analysis of the interstitial (pore) water from the 1000 mg/kg test concentration on Day 28 showed a measured concentration of 7.05 mg/l.

Analysis of the overlying water on Days 0 and 28 showed measured concentrations to be less than the (LOQ) of the analytical method (assessed as 0.49 mg/l). 

It should be noted that the test was designed to evaluate the toxicity of single substances and not complex mixtures such as the test item 'Distillates (Fischer-Tropsch), C8-26-branched and linear' which is considered as an UVCB^[*]substance. This means that greater care should be taken in the interpretation of the data.

[*]UVCB substance: Substance of Unknown or Variable composition, Complex reaction products or Biological materials. (REACH Technical Guidance for Identification and Naming of Substances)

Conclusion

The Day 28 EC₅₀based on nominal test concentrations was was 590 mg/kg with 95% confidence limits of 310 to greater than 1000 mg/kg. The No Observed Effect Concentration was 100 mg/kg and the Lowest Observed Effect Concentration was 320 mg/kg.

Description of key information

- (28d) EC50 and (28d) NOEC for Lumbriculus variegatus (OECD 225): 590 mg/kg sediment dw (nominal, based on: mortalilty) and 100 mg/kg sediment dw (nominal, based on: mortality), respectively [test mat. 'Distillates (Fischer-Tropsch), C8-26-branched and linear'];

- (28d) EC50 and (28d) NOEC for Chironomus riparius (OECD 218): >1000 mg/kg sediment dw (nominal, based on: emergence rate and adult mortality) and  ≥1000 mg/kg sediment dw (nominal, based on: emergence rate and adult mortality), respectively [test mat. 'Distillates (Fischer-Tropsch), C8-26-branched and linear'];

- (28d) EC50 and (28d) NOEC for Chironomus riparius (OECD 218): >1000 mg/kg sediment dw (nominal, based on: development rate) and ≥1000 mg/kg sediment dw (nominal, based on: development rate), respectively [test mat. 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'].

Key value for chemical safety assessment

EC50 or LC50 for freshwater sediment:

590 mg/kg sediment dw

EC10, LC10 or NOEC for freshwater sediment:

100 mg/kg sediment dw

Additional information

Measured toxicity data are not available for 'Paraffin waxes (Fischer-Tropsch), full-range, C15-50 - branched and linear':

The toxicity of the closely related substance GTL Base Oil Distillates (covering the carbon range from C18 to C50) has been determined by Harlan (Goodband, 2011) in a test with the sediment organism Chironomus riparius. The test was conducted in accordance with OECD Test Guideline 218.

Chironomus riparius were exposed to formulated spiked sediment nominal loading rates of 100, 180, 320, 560 and 1000 mg/kg of test material over a period of 28 days. The study was carried out according to GLP and analytical monitoring took place. Analytical work on the ‘wet’ sediment was problematic, therefore analysis on the ‘dry’ sediment was also carried out.

The test results, expressed as the EC₅₀ and NOEC values, showed that the sample was not toxic to sediment organisms at 1000 mg/kg over the time period tested.

Analysis of the ‘dry’ sediment on the day of preparation (Day -7) showed measured concentrations to range from 90% to 111% of nominal. Analysis of the overlying and interstitial pore water on Days 0 and 28 showed measured concentrations to be <LOQ.

The toxicity of the supporting substance GTL Gasoil (covering the carbon range from C8 to C26) has been determined by Harlan Laboratories (Goodband and Mullee, 2011) in a test with the sediment organism Chironomus riparius. The test was conducted in accordance with OECD Test Guideline 218.

Chironomus riparius were exposed to formulated spiked sediment nominal loading rates of 1000 mg/kg of test material over a period of 28 days, following a range finding test where no effects were recorded at 1000 mg/kg. The study was carried out according to GLP and analytical monitoring was performed. Analytical work on the ‘wet’ sediment was problematic, therefore analysis on the ‘dry’ sediment and wet sediment ‘pre-dried’ before analysis sediment was also carried out.

The test results, expressed as the EC₅₀ and NOEC values, showed that the sample was not toxic to sediment organisms at 1000 mg/kg over the time period tested.

Analysis of the ‘dry’ sediment on the day of preparation (Day -7) showed measured concentrations to be 93% of nominal. Analysis of the ‘pre-dried’ sediment showed the measured concentrations to be stable for the duration of the study. Analysis of the overlying and interstitial pore water on Days 0 and 28 gave measured concentrations below the limit of quantitation of the analytical method.

A further test (with the same substance GTL Gasoil, C8-C26) was performed with the sediment organism Lumbriculus variegatus (Goodband and Mullee, 2012). 40-60 worms of Lumbriculus variegatus were exposed to formulated spiked sediment at nominal ‘dry weight’ concentrations of 10, 32, 100, 320 and 1000 mg/kg sediment. The study was carried out according to OECD test guideline 225 and in compliance with GLP. Analytical monitoring was performed, however analysis of ‘wet’ sediment was problematic, therefore measurements with sediment that was ‘pre-dried’ prior to analysis were also carried out.

Analysis of the pre-dried sediment at nominal concentrations of 100, 320 and 1000 mg/kg dw sediment (dried at 60°C for approximately 24 hours) gave measured concentrations of 68% to 78% of nominal and from 28% to 38% nominal on days -9 and 0, respectively. Analysis of the 1000 mg/kg ‘dry’ sediment on Day 28 showed a measured concentration of 51% of nominal. Results for the 10 and 32 mg/kg dw sediment test concentrations were variable since these were close to or below the limit of quantitation of the analytical method. The low recovery rates can be attributed to the difficult nature of the test substance; losses may have occurred due to volatilisation during media preparation, or due to non-extractable residues bound to the sediment phase.

Analysis of the overlying water on Days 0 and 28 gave measured concentrations less than the limit of quantitation of the analytical method. Results for the interstitial pore water were below the limit of quantitation on Days 0 and 28 at 10 mg/kg dw sediment and on Day 0 at 1000 mg/kg dw sediment. A measured interstitial pore water concentration of 7.1 mg/l was determined at 1000 mg/kg dw sediment on Day 28.

Based on survival, the 28-day EC₅₀ was determined to be 590 mg/kg dw sediment, and the NOEC was 100 mg/kg dw sediment. There were no significant differences in body weight compared to controls at any test concentration.

It should be noted that the tests were designed to evaluate the toxicity of single substances and not complex mixtures such as the test items, which are considered as UVCB substances. This means that greater care should be taken in the interpretation of the data.

 

Conclusion:

Long-term (28-d) sediment toxicity tests are available for two GTL-derived substances in the relevant carbon number range. For GTL Gasoil (C8-C26) tests were conducted up to 1000 mg/kg dw loading rate with Lumbriculus variegatus and Chironomus riparius. Based on the studies available, Lumbriculus variegatus appeared to be the more sensitive species, with an EC₅₀ of 590 mg/kg dw and NOEC of 100 mg/kg dw based on mortality. For Chironomus riparius, the EC50 was >1000 mg/kg dw and NOEC ≥1000 mg/kg dw.

For GTL Base Oil Distillates (C18-C50) a study with Chironomus riparius was conducted. In this study, the EC₅₀was >1000 mg/kg dw and NOEC ≥1000 mg/kg dw for development. The EC₅₀ was >1000 mg/kg dw and NOEC = 560 mg/kg dw for mortality and emergence rate.

Finally, the available studies indicate that (saturated) hydrocarbons in the carbon range C8-C50 are not toxic to sediment-dwelling organisms at high dose levels, whereby it is safe to say that the results are also applicable for the substance 'Paraffin waxes (Fischer–Tropsch), full-range, C15–C50 branched and linear'.