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Sediment toxicity

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Reference
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-08-15 to 2018-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)
Version / remarks:
2007
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
SEDIMENT
- Concentrations: all
- Sampling interval: day 0,7 and 28
- Sample storage before analysis: All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

PORE WATER
- Concentrations: all
- Sampling interval: day 0,7 and 28
- Sample storage before analysis: All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

OVERLYING WATER
- Concentrations: all
- Sampling interval: day 0,7 and 28
- Sample storage before analysis: All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.
Vehicle:
yes
Remarks:
acetone
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
The respective test item amount was weighed out for a stock solution and dissolved in Acetone. The stock solution was further diluted with Acetone to receive the respective application solutions. The appropriate amount of spiking solution (2.5 mL per replicate) was thoroughly mixed with a sub-quantity of the artificial sediment (10 g per replicate). After complete evaporation of the solvent, the spiked portion of artificial sediment was thoroughly mixed with the remaining sediment. Demineralised water was added to the artificial sediment to adjust the humidity of the artificial sediment to a moisture of 30 %. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 45 g soil dry weight was filled into test vessels.
Test organisms (species):
Lumbriculus variegatus
Details on test organisms:
TEST ORGANISM
- Breeding conditions: Breeding is performed at the test facility at 20 ± 2 °C and diffuse light (100 - 500 lx, photoperiod 16 h light daily). The dissolved oxygen concentration is > 70 % of the air saturation value corresponding to 6.2 mg O2/L. All worms used in the test originated from the same delivery of the supplier and the same stock.
- Medium: Breeding of L. variegatus is performed on quartz sand in reconstituted water containing 125 mL from each stock solution poured in a can and filled up to 25 L with demineralised water.
- Feeding during breeding: TetraMin® flake food (Tetra) is provided twice per week.
- Feeding during test: Powder of Urtica dioica (stinging nettle, Heinrich Klenk GmbH & Co KG, 97525 Schwebheim, Germany) was mixed into the sediment at the day of application. The amount was 0.4 % of the sediment dry weight.
Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
no
Duration:
28 d
Exposure phase:
total exposure duration
Test temperature:
Room or Water temperature 20 ± 2 °C
pH:
Sediment at start: 6.70
During the test: 6-9
Dissolved oxygen:
Above 70 % of the air saturation value corresponding to 6.2 mg/L O2 (internal quality criterion).
Nominal and measured concentrations:
Nominal test item concentration: control, solvent control, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mg/kg dw
Details on test conditions:
TEST SYSTEM
- Test container: 250 mL glass beakers (6 cm diameter)
- Sediment volume: 45 g dry weight per vessel / ca. 1.0 - 3.0 cm from the ground
- Overlying water volume: per replicate 150 mL
- Depth of sediment and overlying water: Water levels were topped up with demineralised water each working day during the study to compensate losses due to evaporation.
- Aeration: yes, gentle aeration via Pasteur pipettes. Aeration was checked each working day.

EXPOSURE REGIME
- No. of organisms per container (treatment): 10 synchronised worms
- No. of replicates per treatment group: 4 replicates per treatement group, one additional replicate for analytical measurements on day 0, 7, 28 each
- No. of replicates per control / vehicle control: 6 replicates per control, one additional replicate for analytical measurements on day 0, 7, 28 each
- Feeding regime: Powder of Urtica dioica (stinging nettle, Heinrich Klenk GmbH & Co KG, 97525 Schwebheim, Germany) was mixed into the sediment at the day of application. The amount was 0.4 % of the sediment dry weight.

OVERLYING WATER CHARACTERISTCS
- Type of water: demineralised water
- Total organic carbon: 2.23 %

CHARACTERIZATION OF SEDIMENT
Artificial sediment containing:
- 5 % peat, air-dried and finely ground
- 20 % kaolin, with a kaolinite content > 30 %
- 75 % quartz sand (> 50 % particles sized 50-200 pm)
- 0.40 % calcium carbonate (CaCCb) to achieve a pH of 6.70

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark
- Light intensity: 100 - 500 lx

EFFECT PARAMETERS MEASURED:
Visual observations of behavioural differences to the control were recorded three times per week. The endpoints of the study as determined after day 28 were the number of surviving worms per replicate and the total dry biomass of worms per replicate. Worms were considered dead if showing no response to a mechanical stimulus, signs of decomposition or by absence. Living worms were assigned as complete worms or incomplete worms.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Test concentrations and Spacing factor for test concentrations: 3.125 - 6.25 - 12.5 - 25 - 50 - 100 - 200 mg/kg sediment dry weight (factor 2).
Reference substance (positive control):
yes
Remarks:
A reference item study is performed once a year with Potassium chloride. The most recent reference item test was carried out from 2018-03-026 and 2018-04-26.
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
50 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Total number of worms/reproduction
Duration:
28 d
Dose descriptor:
EC50
Remarks:
extrapolated
Effect conc.:
243 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Total number of worms/reproduction
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
100 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Total number of worms/reproduction
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 200 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
200 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Mortality:
No mortality of worms was observed in the controls and all test item concentrations after exposure to the test item over 28 days. Since no statistically significant difference was found between the control and the solvent control, both controls were pooled for further evaluation. The total number of worms and thus the reproduction did not statistically significantly differ compared to the pooled control in the test item concentrations 3.125 to 50 mg/kg sediment dry weight. However, at the test item concentration 100 and 200 mg/kg sediment dry weight the total number of worms and thus the reproduction was statistically significantly reduced.

- Behaviour and pathological Symptoms:
No evident changes in the worm behaviour or pathological symptoms were observed in the control as well as in all test item concentrations during the 28 days of exposure

- Dry Biomass of the Worms per Replicate:
At test start the worms had a body weight of 5.95 mg dry weight/10 worms (mean weight of two determinations). At test end, the mean biomass dry weight of the worms was not statistically significantly different at all test item concentrations compared to the pooled control.

- Measured Exposure Concentrations:
Analytical evaluation of the test item and control was carried out via GC-MS/MS for sediment layer, aqueous phase and pore water on day 0, day 7 and day 28. The measured concentrations of the test item in the sediment were in the ranges from 87% to 112% at day 0, from 29% to 93% at day 7 and from 59% to 153% of the nominal concentrations at day 28. No recoveries of the concentrations of the test item in the aqueous phase and pore water could be determined. Control and solvent control samples were < LOQ, except on day 0 in the control due to carry-over effects within the sample preparation.

- Validity Criteria:
The study was performed according to OECD guideline 225 (2007) and GLP principles. The results met the validity criteria in all points:
- During the test, the oxygen concentration was > 30 % of the air saturation value, corresponding to 2.7 mg/L O2.
- The pH-value was between 6 and 9 in the overlying water during the test.
- In the control group the average number of living worms per replicate increased by a factor of at least 1.8.
Results with reference substance (positive control):
- Results with reference substance valid: yes

Mortality after 28 days of exposure

Test item concentration (mg/kg sediment dry weight) Number of Dead or Missing Worms
Mean Value
Mortality (%)
Control 1 1.70
Solvent control 0 0
3.125 0 0
6.25 0 0
12.5 0 0
25 0 0
50 0 0
100 0 0
200 0 0

Total worm number after 28 days of exposure

Test item concentration (mg/kg sediment dry weight) Number of Surviving Worms
Mean Value
Standard Deviation ± Inhibition to pooled control (%) Sign.*
Control 19.8 5.42 - -
Solvent control 17.2 4.07 - -
Pooled control 18.5 4.78 - -
3.125 13.3 3.59 28.4 no
6.25 15.8 3.59 14.9 no
12.5 16.0 1.41 13.5 no
25 13.5 1.00 27.0 no
50 16.0 2.58 13.5 no
100 11.8 0.50 36.5 yes
200 12.0 0.82 35.1 yes

*= statistically significantly different compared to pooled control (Williams Multiple Sequential t-test procedure, alpha = 0.05)

 

Reproduction after 28 days of exposure

Test item concentration (mg/kg sediment dry weight) Number of Surviving Worms
Mean Value
Standard Deviation ± Inhibition to pooled control (%) Sign.*
Control 9.83 5.42
Solvent control 7.17 4.07
Pooled control 8.50 4.78
3.125 3.25 3.59 61.8 no
6.25 5.75 3.59 32.4 no
12.5 6.00 1.41 29.4 no
25 3.50 1.00 58.8 no
50 6.00 2.58 29.4 no
100 1.75 0.50 79.4 yes
200 2.00 0.82 76.5 yes

*= statistically significantly different compared to pooled control (Williams Multiple Sequential t-test procedure, alpha = 0.05)

Validity criteria fulfilled:
yes
Conclusions:
Based on results of an OECD 225 compliant study with the test item an NOEC(Total number of worms/reproduction) of 50 mg/kg sediment dw was determined as key value.
Executive summary:

The effects of the test item on the oligochaete Lumbriculus variegatus in a water-sediment system were determined. The study was carried out according to OECD Guideline 225 (2007). The test duration was 28 days from the insertion of the test organisms. The study was performed by spiking artificial sediment with the test item concentrations 3.125, 6.25, 12.5, 25, 50, 100 and 200 mg/kg sediment dry weight. A control using untreated artificial sediment as well as a solvent control using artificial sediment treated with the solvent were set up. Six replicates per control and solvent control and four replicates per test item concentration were set up for biological investigations. Water quality parameters (temperature, pH-value, O2-concentration, ammonium and total hardness) were determined throughout the study using an additional replicate for the controls and the test item concentrations, each. Three additional replicates for chemical analysis on day 0, day 7 and day 28 were set up for the controls and each test item concentration. Analytical evaluation of the test item and control was carried out via GC-MS/MS for sediment layer, aqueous phase and pore water on day 0, day 7 and day 28. The measured concentrations of the test item in the sediment were in the ranges from 87% to 112% at day 0, from 29% to 93% at day 7 and from 59% to 153% of the nominal concentrations at day 28. No recoveries of the concentrations of the test item in the aqueous phase and pore water could be determined. Control and solvent control samples were < LOQ, except on day 0 in the control due to carry-over effects within the sample preparation. After 28 days of exposure, it induced no mortality in all test item concentrations. Since no statistically significant difference between the control and the solvent control was determined, both controls were pooled for further evaluation.

The total number of worms and thus the reproduction did not statistically significantly differ compared to the pooled control in the test item concentrations 3.125 to 50 mg/kg sediment dry weight. However, at the test item concentration 100 and 200 mg/kg sediment dry weight the total number of worms and thus the reproduction was statistically significantly reduced.All validity criteria were fulfilled.

Description of key information

Based on results of an OECD 225 compliant study with the test item an NOEC (Total number of worms/reproduction) of 50 mg/kg sediment dw was determined as key value.

Key value for chemical safety assessment

EC50 or LC50 for freshwater sediment:
243 mg/kg sediment dw
EC10, LC10 or NOEC for freshwater sediment:
50 mg/kg sediment dw

Additional information

The effects of the test item on the oligochaete Lumbriculus variegatus in a water-sediment system were determined. The study was carried out according to OECD Guideline 225 (2007). The test duration was 28 days from the insertion of the test organisms. The study was performed by spiking artificial sediment with the test item concentrations 3.125, 6.25, 12.5, 25, 50, 100 and 200 mg/kg sediment dry weight. A control using untreated artificial sediment as well as a solvent control using artificial sediment treated with the solvent were set up. Six replicates per control and solvent control and four replicates per test item concentration were set up for biological investigations. Water quality parameters (temperature, pH-value, O2-concentration, ammonium and total hardness) were determined throughout the study using an additional replicate for the controls and the test item concentrations, each. Three additional replicates for chemical analysis on day 0, day 7 and day 28 were set up for the controls and each test item concentration. Analytical evaluation of the test item and control was carried out via GC-MS/MS for sediment layer, aqueous phase and pore water on day 0, day 7 and day 28. The measured concentrations of the test item in the sediment were in the ranges from 87% to 112% at day 0, from 29% to 93% at day 7 and from 59% to 153% of the nominal concentrations at day 28. No recoveries of the concentrations of the test item in the aqueous phase and pore water could be determined. Control and solvent control samples were < LOQ, except on day 0 in the control due to carry-over effects within the sample preparation. After 28 days of exposure, it induced no mortality in all test item concentrations. Since no statistically significant difference between the control and the solvent control was determined, both controls were pooled for further evaluation.

The total number of worms and thus the reproduction did not statistically significantly differ compared to the pooled control in the test item concentrations 3.125 to 50 mg/kg sediment dry weight. However, at the test item concentration 100 and 200 mg/kg sediment dry weight the total number of worms and thus the reproduction was statistically significantly reduced. All validity criteria were fulfilled.