Registration Dossier

Administrative data

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: The radiolabeled test item was applied to the sediment in static water-sediment systems.
Details on test material:
Radiolabeled Test Item:
Identity: [C-14] Dioctylfumarate
Test Item Name for Report: Dioctylfumarate
Batch: 12BLY061
Harlan Laboratories Internal Logistic No.: 256545/A
Radiochemical Purity: >97% (re-determined before use)
Specific Activity: 3,48 MBq/mg
Expiry Date: Not needed since the purity was determined by Harlan Laboratories Ltd. before use.
Storage Conditions (Sponsor): For long term, storage at - 80 °C
Storage Conditions (Harlan Laboratories): - 80 °C
Stability of Test Item in Vehicle: Was determined before and after application.
Safety Precautions: Routine hygienic procedures according to the Swiss Legislation on Radiological Protection (Switzerland, Ordinance of June 22, 1994).

Unlabeled Test Item:
Identity: Dioctylfumarate
Batch No.: LEDF1C7047
CAS Number: 141-02-6
Molecular Formula: C20H36O4
Purity: 99.2%
Molecular Weight: 340.56 g/mol
Solubility in Water at 20°C: 0.00119 g/L
Expiration Date: 30 November 2014
Storage Conditions (as provided by the Sponsor): not to exceed room temperature (max. 25°C) during storage over a longer period of time.
Storage Conditions (as handled at Harlan Laboratories): At room temperature at about 20 °C, in the dark.
Safety Precautions: Routine hygienic procedures are sufficient to ensure personnel health and safety.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The sampling for the analysis of the test item concentrations during the definitive test is described below and additionally shown in the sampling scheme.
Before the test item application, samples were taken from the highest application solution. For the determination of the stability of the test item in the application solutions, a sample was taken also immediately after the application from the application solution of the highest test concentration.
The analytical samples from day 0 and 6 were taken from test beakers prepared exclusively for analytical measurements in parallel to the biological test (two test beakers were prepared for each sampled test concentration, and one for the solvent control). One of the two treated test beakers and the solvent control beaker were sampled on day 0. The other treated replicates were incubated under the conditions of the test until sampling on day 6. To these additional replicates, test organisms were inserted and food was added as in the biological part of the study. On day 28, analytical samples were taken from the test beakers in the biological test.
On each sampling date, the water samples were taken from the water column without previously mixing the water.
For the determination of the test item concentrations in the pore water and sediment, the water column was siphoned from the test beaker.
The pore water was obtained by centrifugation of the wet sediment at about 10'000 g for 30 minutes.
The resultant overlaying water is defined as pore water. This pore water was sampled after recording the total pore water volume of each sediment.
Then the sediment sample was taken for analysis (without pore water).

Test substrate

Vehicle:
yes
Details on sediment and application:
Glass beakers (600 mL, approximately 8 cm in diameter) were used as test vessels. Each test beaker was covered with a lid containing a mosquito net to prevent the escape of emerged adult test animals. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.
An artificial (formulated) sediment was used according to the test guideline. The artificial sediment was prepared on the basis of dry weights as follows:

% distribution
- Sphagnum peat: 5%
(air dried, very finely ground to ≦1 mm)
organic carbon content 47.3%)
- Kaolin clay (content of Al2O3: 35.0%): 20%
- Sand (Sihelco 36): 75%
Size analysis
0.25 mm 1.0%
0.18 mm 7.0%
0.125 mm 80%
0.09 mm 11%
<0.09 mm 1.0%
- Calcium carbonate (CaCO3): 0.35%

The total organic carbon (TOC) in this artificial sediment was 2.4% (based on dry weight).
All dry constituents were weighed to the correct portions. The finely ground peat was moistened with purified water by intense mixing using an ultra-turrax and gentle stirring for four days at room temperature in the dark. The pH of the peat suspension was 5.6 at start of moistening. CaCO3 was added for pH adjustment, and after the moistening procedure the pH was 6.3. Then all constituents were thoroughly blended in a HOBART laboratory mixer to obtain a homogeneous wet sediment. Purified water was added to obtain sufficient moisture of the final mixture (46 %, based on dry weight).
A sediment sample of about 10 g was shaken with 25 mL of 1M KCl solution for 30 minutes and then the pH was measured in this suspension. The pH of the final mixture of sediment was 7.2.
The artificial sediment was checked for absence of chemical contamination under Harlan Laboratories study D61982. The following parameters were analyzed in a representative sample of the artificial sediment: heavy metals (cadmium, arsenic, lead, mercury and copper), pesticides (lindane, heptachlor, malathion, total DDT, dieldrin), and PCBs (28, 52, 101, 138, 153, 180).
Since the test item is not sufficiently soluble in water, stock/application solutions were prepared in the volatile solvent chloroform. The application solutions of the lower test concentrations were prepared by diluting the stock solution or the application solution of the highest test concentration by the solvent. In this way defined volumes of these application solutions (1 mL) were mixed with 10 g of sand for each test beaker. After completely evaporating the solvent, the sand/test item mixture was thoroughly mixed with the remaining part of wet sediment of each single test beaker by means of a roller mixer in a mixing flask. The amount of sand necessary for the preparation of the sand/test item mixtures was taken into account when preparing the sediments for the test beakers.
The application solutions were prepared immediately before spiking the sediment. The actual concentrations in the application solutions were analytically verified by LSC measurements, and the purity of the radiolabeled test item by HPLC analysis.
The sediments of the solvent control were prepared like the spiked sediments including application of the solvent, but without test item. The sediments of the control were prepared like the spiked sediments, but without solvent and test item.
The wet artificial sediment was filled into each test vessel at a layer of approximately 1.5 cm depth. This amount corresponded to 130 g wet weight with 46% water content (or 89 g dry sediment). Before adding the test water, the sediment surface was covered with a plastic plate, which floated as the water was poured onto it. Then 240 mL of test water were poured into each beaker very slowly, taking care not to disturb the sediment. The total water volume per beaker was 250 mL (corrected for 10 mL of the application solution used for spiking the test item into the sediment) and corresponded to a water column of 6 cm depth. Thus, the ratio of the depth of the sediment layer to the depth of the overlying water was 1:4. After filling the vessels with water, the plastic plate was removed.
The vessels were prepared two days before inserting the test animals, and were incubated during this period under the conditions of the test.
The water level was marked outside on the test vessels. Water levels did not change by more than 10% during the test period. If necessary, purified water was filled up to the normal water level (15 mL per beaker on Day 18).
The test concentrations were calculated as concentrations of the test item in the dry sediment. The following five nominal test concentrations of Dioctylfumarate were tested: 10, 32, 100, 320 and 1000 mg/kg dry sediment.
The route of administration of the test item was chosen in compliance with the test guideline. The test concentrations were based on the results of a prolonged range-finding toxicity test with larvae of Chironomus riparius (GLP).
In parallel to the treatments, a non-treatment control and a solvent control were was tested.

Test organisms

Test organisms (species):
Chironomus riparius
Details on test organisms:
The study was performed with larvae of the midge Chironomus riparius bred at Harlan Laboratories Ltd. The breeding is conducted under similar conditions (temperature, light, test water) as used in the test.
Only fresh egg masses were used as source for the test animals. At the date when the test animals were placed into the test beakers, the larvae were 2–3 days old (first-instar larvae).
The midge Chironomus riparius is a preferred freshwater aquatic insect species used to evaluate the toxicity of chemicals present in sediments. The test method and the test species are recommended by the international test guidelines.
Six days before inserting the larvae into the test beakers, some fresh egg masses were taken from the test organism culture and deposited into small vessels in test water with a small amount of food (mixture of fresh green algae Scenedesmus subspicatus from a laboratory culture and a Tetra Min fish food suspension).
Twenty larvae of the first larval stage (2–3 days old) were allocated randomly to each test vessel by means of a suitable pipette (four collectives of five larvae each, per vessel). When adding the larvae and also for the following 24 hours, the aeration of the water in the test vessels was stopped.
The larvae were inserted into the test vessels two days after spiking the sediment and establishing the water-sediment systems. The day of the larvae insertion was defined as day 0 of the study.

Study design

Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
no
Exposure duration
Duration:
28 d
Exposure phase:
total exposure duration
Remarks:
28 days after introduction of the larvae (5 days after emergence of the last test animal in the controls).

Test conditions

Hardness:
Water hardness was measured by titration (Aquamerck, No. 1.08039.0001)
Day 0: Water hardness 3.1 mmol/L for control, solvent control, and vessel with 1000 mg/kg
Day 28: Water hardness: control 3.7 mmol/L; solvent control: 3.7 mmol/L, vessel with 1000 m/kg: 3.8 mmol/L
Test temperature:
Water temperature: 19.5-20.5 °C during the experiment. The water temperature differed by less than 1.0 °C between beakers at any time during the test.
pH:
During the test period, the pH values in the test media ranged from 7.8 to 8.5.
Dissolved oxygen:
> 7.2 mg/L ( 78 % saturation) throughout the test period
Ammonia:
Ammonium was measured by photometrical measurement (Merck Spectroquant No. 1.14558.0001).
Day 0: Ammonia concentration <0.20 mg/L for control, solvent control, and vessel with 1000 mg/kg
Day 28: Ammonia concentration: control 1.27 mg/L; solvent control: 3.80 mg/L, vessel with 1000 m/kg: 1.10 mg/L
Nominal and measured concentrations:
The following five nominal test concentrations of Dioctylfumarate were tested: 10, 32, 100, 320 and 1000 mg/kg dry sediment.
Details on test conditions:
Light conditions: A 16-hour light to 8-hour dark photoperiod with a 30 minute transition period between light and darkness. Light intensity during the light period was within the range of approximately 680–980 Lux (measured approximately at water surfaces of the test beakers).

Aeration: During the whole study (with exception of the period of 24 hours after the introduction of the larvae) the water in the water-sediment systems was gently aerated through a glass Pasteur pipette, fixed above the sediment layer.

Four replicates (test beakers) were tested in the biological test at each test concentration and in the control and solvent control. Additional replicates were prepared in parallel for the analytical requirements .


The number of emerged adults and their sex were recorded daily from Day 10 after larval insertion until Day 28 (5 days after emergence of the last test animals in the controls). After sex identification the midges were removed from the vessels and discarded. Only the number of fully emerged male and female midges was counted. The number of visible pupae that failed to emerge was counted separately in each test beaker. Any other signs of intoxication of the larvae, pupae and emerged midges were recorded. The test vessels were searched for deposited egg masses to prevent re-introduction of new larvae into the sediment.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
320 mg/kg sediment dw
Nominal / measured:
nominal
Basis for effect:
other: the emergence ratios, development rates of Chironomus riparius, and observations on signs of intoxication
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
1 000 mg/kg sediment dw
Nominal / measured:
nominal
Basis for effect:
development rate
Remarks:
slightly reduced
Details on results:
Emergence ratio
The numbers of fully emerged midges per test vessel on each inspection day: The values are listed separately for males, females and for pooled sexes. The emergence ratios per vessel in the controls ranged from 70 to 95 % (thus fulfilling the guideline validity criterion of ≥ 70 %). The mean emergence ratio of the midges in the solvent control was not statistically significantly different from the control (results of a Student-t test with the arcsin-transformed emergence ratios, alpha = 0.05, two-sided).
The calculated mean emergence ratios for pooled sexes: Up to and including the highest nominal test concentration of initial 1000 mg dioctylfumarate/kg dry sediment, the mean emergence ratios of pooled sexes were not statistically significantly lower than in the solvent control (results of a Welch t-test with the arcsin-transformed emergence rates, alpha = 0.05, one-sided smaller).
The 28-day EC15 and the 28-day EC50 for the arcsin-transformed emergence rate of pooled sexes could not be calculated due to the low toxicity of the test item. Both values are, therefore, > 1000 mg/kg dry sediment.
The sex of the small inserted larvae at the start of the test can not be differentiated. Thus, a separated emergence ratio for males and females can not be determined, and is, therefore, not taken into account.

Development rate
The calculated mean development rates for both sexes: The midges in the control and solvent control had emerged between days 13 and 23 (and thus, fulfilled the validity criterion of the test guideline requesting the emergence not earlier than on day 12 and not later than on day 23). The mean development rate of the male midges in the solvent control was not statistically significantly different from the control (results of a Student's t-test, alpha = 0.05, two-sided). The mean development rate of the female midges in the solvent control was statistically significantly different from the control (results of a Student's t-test, alpha = 0.05, two-sided). Since this statistically significant difference is within 10 % of variance between control and solvent control and can be relayed to single replicates and also to few individual female midges, this statistically significant effect is not seen as biologically relevant. Based on the statistically significant difference, both controls were pooled for calculation of NOEC concentration for the development rate of the female midges.
Up to and including the test concentration of initial 320 mg/kg dry sediment, the mean development rates of males and females were not statistically significantly lower than in the solvent control (results of a Williams t-test, alpha = 0.05, one-sided smaller). At the highest nominal test concentration of initial 1000 mg/kg dry sediment, the mean development rates of both males and females were slightly, but statistically significantly reduced compared to the solvent control.
The 28-day EC15 and the 28-day EC50 for the development rate of males and females could not be calculated due to low toxicity of the test item. Both values are, therefore, > 1000 mg/kg dry sediment.
*Symptoms of toxicity
No symptoms of toxicity were observed at the larvae, pupae and emerged midges during the study.

Any other information on results incl. tables

The measured test item concentrations in the test media one hour after application corresponded well with the initial nominal test concentrations. The variability between the analytical results of the parallel test vessels per treatment was very low. Therefore, all reported biological results are related to the nominal initial concentrations of Dioctylfumarate in the dry sediment.

At both measured test concentrations of 100 and 1000 mg/kg dry sediment the radioactive residue in the overlaying water columns remained nearly constant at a low level throughout the whole test period from 0.9 and 0.5 % at test start to 1.4 and 0.5 % on day 6 of the nominal values after application to 1.2 and 0.2 % of the initial values at the test termination after 28 days.

At both measured test concentrations of 100 and 1000 mg/kg dry sediment the radioactive residue in the pore water remained nearly constant at a low level throughout the whole test period from 0.6 and 0.3 % at test start to 1.5 and 0.4 % on day 6 of the nominal values after application to 1.1 and 0.5 % of the initial values at the test termination after 28 days.

The radioactivity in the sediments continuously decreased during the test period in both measured concentrations of 100 and 1000 mg/kg dry sediment from 96.6 and 97.1 % at test start to 92.6 and 92.8 % on day 6 to 60.0 and 81.9 % of the initial values at the test termination after 28 days.

A part of the radioactivity (between 2.8 and 10.6 % in both measured concentrations throughout the test period) was bound to sediment and was not extractable. The not extractable amount of radioactivity increased over time.

No metabolites of the test item were found on days 0 and 28 of the study, whereas three metabolites overall were found on day 6 of the study, all in minor amounts (< 5.5 % of applied test item).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The overall 28-day NOEC of dioctylfumarate for Chironomus riparius in this sediment spiked study was 320 mg/kg dry sediment. The overall 28 day LOEC was 1000 mg/kg dry sediment due to a slightly, but statistically significantly reduced development rate of the female and male midges.
Executive summary:

Toxic effects of the test item dioctylfumarate on the development of sediment-dwelling larvae of the midge Chironomus riparius in water-sediment systems were investigated following the OECD guideline 218 (sediment-water chironomid toxicity test using spiked sediment, 2004).

First-instar larvae of Chironomus riparius were exposed for a period of 28 days until full maturation of the larvae to adult midges. The test parameters of the study were development time/rate of the midges and emergence ratio as the number of fully emerged male and female midges.

The radiolabeled test item was applied to the sediment in static water-sediment systems. The nominal initial test item concentrations in the sediments were 10, 32, 100, 320 and 1000 mg dioctylfumarate per kg dry sediment. A control (water-sediment systems without test item application) and a solvent control group were tested in parallel. The spiked sediments were kept for two days under the test conditions to reach equilibrium between sediment and aqueous phases as pore water and overlying water before the introduction of the first-instar larvae of Chironomus riparius.

The measured test item concentrations in the test media one hour after application corresponded well with the nominal initial test concentrations. The variability between the analytical results of the parallel test vessels per treatment was very low. Therefore, all reported biological results are related to the nominal initial concentrations of dioctylfumarate in the dry sediment.

At both measured test concentrations of 100 and 1000 mg/kg dry sediment the radioactive residue in the overlaying water columns remained nearly constant at a low level. Throughout the whole test period, the detected values ranged from 0.9 and 0.5 % at test start to 1.4 and 0.5 % on day 6 of the nominal values after application to 1.2 and 0.2 % of the initial values at the test termination after 28 days.

At both measured test concentrations of 100 and 1000 mg/kg dry sediment the radioactive residue in the pore water remained nearly constant at a low level throughout the whole test period from 0.6 and 0.3 % at test start to 1.5 and 0.4 % on day 6 of the nominal values after application to 1.1 and 0.5 % of the initial values at the test termination after 28 days.

The radioactivity in the sediments continuously decreased during the test period in both measured concentrations of 100 and 1000 mg/kg dry sediment from 96.6 and 97.1 % at test start to 92.6 and 92.8 % on day 6 to 60.0 and 81.9 % of the initial values at the test termination after 28 days.

A part of the radioactivity (between 2.8 and 10.6 % in both measured concentrations throughout the test period) was bound to sediment and was not extractable. The non-extractable amount of radioactivity increased over time.

No metabolites of the test item were found on days 0 and 28 of the study, whereas three metabolites overall were found on day 6 of the study, all in minor amounts (< 5.5 % of applied test item).

The biological test results (based on the nominal initial test concentrations calculated as concentrations of the test item in dry sediment) were as follows: 

Results after 28-days

(mg/kg dry sediment)

Emergence rate
(arcsin-transformed)
of pooled sexes
(mg/kg dry sediment)

Development rate
(mg/kg dry sediment)

Males

Females

EC15:

>1000

>1000

>1000

95% confidence limits:

n.d.

n.d.

n.d.

EC50:

>1000

>1000

>1000

95% confidence limits:

n.d.

n.d.

n.d.

NOEC:

1000

320

320

LOEC:

>1000

1000

1000

n.d.: could not be determined due to low toxicity of the test item.

 

Thus, the overall 28-day NOEC of dioctylfumarate for Chironomus riparius in this sediment spiked study was 320 mg/kg dry sediment. The overall 28-day LOEC was 1000 mg/kg dry sediment due to a slightly, but statistically significantly reduced development rate of the female and male midges.