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Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15/04/2005 - 02/12/2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to methodology comparable to OECD TG 218 in compliance with GLP, without deviations that influence the quality of the results. Includes analysis of actual concentrations.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)
Deviations:
yes
Remarks:
The study was executed with Lumbriculus. The analytical investigations were outsourced to a specialised analytical laboratory for analysis and reported separately (Belfroid & Blok, 2006).
Qualifier:
equivalent or similar to
Guideline:
other: Phipps G.L., Ankley G.T., Benoit D.A. & Mattson V.R., 1993. Use of the aquatic oligochaete Lumbriculus variegatus for assessing the toxicity and bioaccumulation of sediment-associated contaminants. Environ.Toxicol.Chem. 12, 269 - 279.
GLP compliance:
yes
Remarks:
GLP Statement included
Analytical monitoring:
yes
Details on sampling:
SAMPLING PROCEDURE
Samples of sediment, pore water and overlying water were taken during the exposure up to 28 days. The overlying water was taken directly from the test vessels by carefully decanting into a glass beaker. The wet sediment was centrifuged at 9425 x g. The pore water was carefully decanted into glass beakers. All samples were deep-frozen at -18°C to -25°C.
- Sampling interval: directly after spiking (day -7), at the end of equilibration (day 0), on day 14 of exposure and at the end of the exposure period (day 28)

SEDIMENT
- Concentrations: 1.2, 3.7, 11.1, 33.3 and 100 mg/kg sediment dry weight (nominal)
- Sample handling: Sediment samples were defrosted, homogenized and a subsample was taken for dry weight determination as described by Egeler et al (2005a). The remaining subsample was freeze-dried. With every run of freeze-drying, a blank sample consisting of 1 g of silicagel was included. A subsample of 2 – 3 g of dried material was collected for shipment. The recovery standard D3-AHTN was added to both freeze dried sediment and blank silicagel.
- Sample storage before analysis: deep-frozen at -18 to -25 degrees Celsius

PORE WATER
- Sample Handling: Frozen samples of overlying water and pore water were defrosted at room temperature. The recovery standard D3-AHTN (deuterated AHTN) was added and the samples were extracted with Speedisks as described by Egeler et al (2005a) and frozen again for shipment
- Sample storage before analysis: deep-frozen at -18 to -25 degrees Celsius

OVERLYING WATER
- Concentrations: 41, 122, 367, 1101 and 3304 mg/l (nominal)
- Sample handling and storage as for pore water
Vehicle:
yes
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: Artificial sediment was formulated. The sediment was amended prior to the application of the test item with finely ground leaves of stinging nettle (Urtica sp.; urtica powder) and cellulose (α-cellulose powder) to an amount of 0.4 - 0.5% of sediment dry weight to ensure that the worms will reproduce under control conditions. In this way it is ensured that the test animals were exposed through the water and sediment as well as by their food.
- Method of mixing: no data
- Details of spiking: The test solutions for application of test item into the sediment were obtained by diluting the stock solution with acetone. From these test solutions an appropriate volume was mixed with an appropriate amount of quartz sand for each treatment (10 g of quartz sand per replicate). The acetone was evaporated completely before the sediment of each concentration level was prepared. The amount of sand provided by the test-item-and-sand mixture was taken into account when preparing the formulated sediment. To ensure that the test item added to sediment was evenly distributed within the sediment, the bulk formulated sediments were thoroughly mixed. From these bulk concentration levels, the sediment was distributed to the individual replicates of each concentration level.
- Equilibration time: 7 days
- Equilibration conditions: same as during test
- Controls: yes, blanks and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 100% in stock solution,
- Evaporation of vehicle before use:


Test organisms (species):
Lumbriculus variegatus
Details on test organisms:
TEST ORGANISM
- Common name: Lumbriculus variegatus
- Strain/clone: Muller
- Source: ECT Oekotoxikologie GmbH; originally from Fischfutter Etzbach (D-53894 Mechemich-Bergheim, Germany)
- Breeding conditions: 20 +/- 2 degrees Celsius
- Feeding during test: feed in sediment (urtica powder and cellulose)

ACCLIMATION
- Acclimation period: 14 days
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
Hardness:
317.9 - 367.9 mg/l CaCO3 (17.8 - 20.6 dH)
Test temperature:
20.8 - 22.0 degrees Celsius
pH:
8.2 - 8.4
Dissolved oxygen:
6.8 - 8.7 mg/l
Ammonia:
1.95 - 4.47 mg/l
Nominal and measured concentrations:
Sediment (nominal): 1.2, 3.7, 11.1, 33.3 and 100 mg/kg sediment dry weight
Overlying water (nominal): 41, 122, 367, 1101 and 3304 mg/l
Actual concentrations were measured in the middle (C3) and highest (C5) concentrations, in sediment, overlying water and porewater on days 0, 14 and 28. The results of the analyses showed that the concentrations of OTNE decreased gradually (from day -7 to day 0), but stayed above 80% of theconcentration at the start of the exposure in almost all tests (between day 0 and day 28). Only in the toxicity test with Lumbriculus the lowest exposure concentration of 4.5 mg/kg dw fell to less than 80% of the concentration at the start of the exposure (C[t=0]). However a similar initial concentration in the Hyalella test was regarded as duplo determination for this Lumbriculus test. The level of the NOEC (33.3 mg/kg, C4) was not included in the samples, but the actual concentration was derived based on the average decrease in the concentrations in C3 and C5. Thus the NOEC was 17.1 mg/kg dw (actual concentration).
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 250 ml glass vessels for biological measurements, 1l vessels for analytical measurements
- Sediment volume: 80 or 200 g
- Overlying water volume: 190 or 550 ml
- Depth: 1.8 - 2.0 cm
- Aeration: yes
- Aeration frequency and intensity: gentle aeration, 2 - 4 bubbles/s, 2- 3 cm above sediment
- Replacement of evaporated test water, if any: yes


EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control / vehicle control: 4 / 6
- Feeding regime: feed in sediment
- Type and preparation of food: cellulose and urtica powder

OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): reconstituted (according to OECD 203), containing:
CaCl2.2H2O (294.0 mg/l), MgSO4.7H2O (123.0 mg/l), NaHCO3 (64.8 mg/l) and KCl (5.75 mg/l)
- Conductivity: 547 uS/cm

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- % dry weight of sphagnum moss peat: 5 ± 0.5%
- Particle size distribution
- % sand: 75-76%
- % clay: 20 ± 1%
- Composition (if artificial substrate):
Constituent % of sediment dry weight
Peat 5 ± 0.5
Quartz sand 75 - 76*
Kaolinite clay 20 ± 1
Urtica powder 0.2 - 0.25
Cellulose powder 0.2 - 0.25
Organic carbon 2 ± 0.5
Calcium carbonate 0.05 - 1
Deionised Water 30 - 50
*The sediment was prepared with less sand to account for the amount of sand used for spiking the test item.

- Method of preparation (if artificial substrate): A suspension of the required amounts of peat in deionised water was prepared using a high-performance homogenising device. The pH of this suspension was adjusted to 5.5 ± 0.5 with CaCO3. The suspension was conditioned for two days with gentle stirring at room temperature, to stabilise pH and establish a stable microbial component. pH was measured again and was adjusted to 6.0 ± 0.5. Then the peat suspension was mixed with the other constituents (sand and kaolinite clay) and deionised water to obtain a homogeneous sediment with a water content in a range of 30-50 per cent of dry weight of the sediment
- Moisture: 34.8 ± 0.2


OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 488 lx


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality, reproduction, dry weight


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study yes, non-GLP
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
17.1 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
33.3 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
33.3 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: total biomass
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
96.6 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
133.3 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: total biomass
Details on results:
- Mortality of test animals at end of exposure period: 13 - 46 surviving worms per vessel
- Behavioural abnormalities: none observed
Reported statistics and error estimates:
In order to estimate mortalities, the numbers of worms that did not react to a gentle stimulus or showed signs of decomposition were considered dead. In cases where the number of worms found on day 28 of the exposure period was less than the number of worms introduced to the vessel, the number of missing worms was added to the number of worms observed dead. In these cases, survival is defined as the difference between the number of worms introduced and the sum of dead and missing worms.

To determine significant differences between the controls (control and solvent control) the replicates of each control were tested for normal-distribution (Kolmogorov-Smirnov-test) and for homogeneity of variances (Cochran's test); thereafter a pairwise comparison test (Student-t Test or Welch T-test) was used. If these tests detected no significant differences, all control and solvent control replicates were pooled.

Normal-distribution of data was tested with the Kolmogorov-Smirnov goodness-of-fit test (two-sided, p ≤ 0.05). Cochran's test (two-sided, p ≤ 0.05) was used to test variance homogeneity. One-way Analysis of Variance (ANOVA) and subsequent multi-comparison tests (Dunnett's Test; one-sided, smaller) were used to calculate whether there were significant differences (p ≤ 0.05) between the controls and the various test item concentrations.

The ECx values were calculated using Probit analysis (Finney, 1971) and maximum likelihood regression.

The statistical software packages ToxRat Professional 2.09 was used for these calculations.
Validity criteria fulfilled:
yes
Remarks:
pH and temperature constant, oxygen saturation >60%, number of worms at end of test increased >20% by the end of the test, emergence: not applicable
Conclusions:
The 28d-NOEC and 28d-EC50 OTNE towards the reproduction of Lumbriculus variegatus are (nominal) 33.3 and 96.6 mg/kg sediment dry weight, respectively. The 28d-NOEC expressed on the basis of the actual concentration was 17.1 mg/kg dw (with total organic carbon content 2.29%).
Executive summary:

The chronic toxicity of OTNE towards Lumbriculus variegatus in sediment was investigated according to methodology comparable to OECD Guideline 218 under GLP. Validity criteria for the test were met. Organisms were exposed to nominal concentrations of 1.2, 3.7, 11.1, 33.3 and 100 mg/kg sediment dry weight. The actual concentrations in the sediment upon introduction of the test organisms were 52% of nominal. On day 14 the measured concentrations were within 80% of the concentrations on day 0. This was also the case for the highest concentration on day 28, but middle concentration fell below 80% (Belfroid and Blok 2006). Survival was not affected up to the highest test concentration of 100 mg/kg dw. Reproduction and growth were not affected up to 33 mg/kg dw. The reproduction was inhibited by 55% and growth was inhibited by 35% at 100 mg/kg (Egeler and Gilberg 2005). After correction for the actual concentration on day 0, the actual NOEC was 17.1 mg/kg dw (Belfroid and Blok 2006).

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15/04/2005 - 31/10/2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to methodology comparable to OECD TG 218 in compliance with GLP, without deviations that influence the quality of the results. Includes analysis of actual concentrations.
Qualifier:
according to
Guideline:
OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)
Deviations:
yes
Remarks:
The analytical investigations were outsourced to a specialised analytical laboratory for analysis and reported separately (Belfroid & Blok, 2006). In the solvent controls, one larva emerged on day 25 after addition to the test vessels (required: day 23).
GLP compliance:
yes
Remarks:
GLP Statement included
Analytical monitoring:
yes
Details on sampling:
SAMPLING PROCEDURE
Samples of sediment, pore water and overlying water were taken during the exposure up to 28 days. The overlying water was taken directly from the test vessels by carefully decanting into a glass beaker. The wet sediment was centrifuged at 9425 x g. The pore water was carefully decanted into glass beakers. All samples were deep-frozen at -18°C to -25°C.
- Sampling interval: directly after spiking (day -7), at the end of equilibration (day 0), on day 14 of exposure and at the end of the exposure period (day 28)

SEDIMENT
- Concentrations: 50, 100, 200, 400 and 1000 mg/kg sediment dry weight (nominal)
- Sample handling: Sediment samples were defrosted, homogenized and a subsample was taken for dry weight determination as described by Egeler et al (2005a). The remaining subsample was freeze-dried. With every run of freeze-drying, a blank sample consisting of 1 g of silicagel was included. A subsample of 2 – 3 g of dried material was collected for shipment. The recovery standard D3-AHTN was added to both freeze dried sediment and blank silicagel.
- Sample storage before analysis: deep-frozen at -18 to -25 degrees Celsius

PORE WATER
- Sample Handling: Frozen samples of overlying water and pore water were defrosted at room temperature. The recovery standard D3-AHTN (deuterated AHTN) was added and the samples were extracted with Speedisks as described by Egeler et al (2005a) and frozen again for shipment
- Sample storage before analysis: deep-frozen at -18 to -25 degrees Celsius

OVERLYING WATER
- Concentrations: 4147, 8295, 16589, 33179 and 82947 mg/l (nominal)
- Sample handling and storage as for pore water
Vehicle:
yes
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: Artificial sediment was formulated according to OECD guideline No. 218 (OECD 2004); peat content 5% of sediment d.w.; addition of Urtica-powder (0.4 - 0.5% on dry sediment) before application of test item to ensure that the larvae grow and emerge
under control conditions and to assure that exposure was both through the sediment and through the food.; no additional feeding.
- Method of mixing: no data
- Details of spiking: test item dissolved in acetone; test item coated on quartz sand, then mixed with wet sediment. The acetone was evaporated completely before the sediment of each concentration level was prepared. The amount of sand provided by the test-item-and-sand mixture was taken into account when preparing the formulated sediment. To ensure that the test item added to sediment was evenly distributed within the sediment, the bulk formulated sediments were thoroughly mixed. From these bulk concentration levels, the sediment was distributed to the individual replicates of each concentration level.
- Equilibration time: 7 days
- Equilibration conditions: same as during test
- Controls: yes, blanks and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 100% in stock solution,
- Evaporation of vehicle before use:


Test organisms (species):
Chironomus riparius
Details on test organisms:
The test system used in this study was Chironomus riparius (Meigen) from in-house cultures, originally supplied by Aventis, D-65962 Frankfurt am Main
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
Hardness:
410.8 - 467.9 mg/L as CaCO3. ( 23.0 - 26.2 °dH )
Test temperature:
19.4 - 20.8 oC
pH:
8.0 - 8.7
Dissolved oxygen:
6.8 - 9.1 mg/L
Ammonia:
0.2 - 10.0 NH4+ mg/L
Nominal and measured concentrations:
Sediment (nominal): 50, 100, 200, 400 and 1000 mg/kg sediment dry weight
Overlying water (nominal): 4147, 8295, 16589, 33179 and 82947 mg/l
Actual concentrations were measured in the middle (C3) and highest (C5) concentrations, in sediment, overlying water and porewater on days 0, 14 and 28. The results of the analyses showed that the concentrations of OTNE decreased gradually (from day -7 to day 0), but stayed above 80% of theconcentration at the start of the exposure (between day 0 and day 28). The level of the NOEC (200 mg/kg, C3, nominal) was included in the samples, actual concentration is 133 mg/kg dw.
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
102 mg/kg sediment dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Based on additional observations: complete emergence, ability to fly and survival after emergence
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
200 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Based on additional observations: complete emergence, ability to fly and survival after emergence
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
400 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
emergence rate
Remarks:
males and females pooled
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:
development rate
Remarks:
males and females pooled
Duration:
28 d
Dose descriptor:
other: EC15
Effect conc.:
486 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
emergence rate
Remarks:
males and females pooled
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
642 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
emergence rate
Remarks:
males and females pooled
Details on results:
Additional observations indicated that at concentrations ≥ 400 mg/kg (d.w.) an increasing percentage of fully emerged midges were dead and/or not able to fly (ratite). Also the percentage of dead, incompletely emerged midges increased at 400 - 1000 mg/kg. These observations were not evaluated statistically. However, since complete emergence, ability to fly, and survival after emergence are relevant for the successful continuity of a population, 200 mg kg-1 sediment dry weight (nominal concentration) is suggested as the overall NOEC to be derived from this study. Expressed as actual concentraion this NOEC is 102 mg/kg sediment dw.

Results are based on statistical evaluation of biological results and nominal concentrations in mg kg-1sediment dry weight.

Additional observations indicated that at concentrations ≥ 400 mg/kg(d.w.) an increasing percentage of fully emerged midges were dead and/or not able to fly (ratite). Also the percentage of dead, incompletely emerged midges increased at 400 - 1000 mg/kg. These observations were not evaluated statistically. However, since complete emergence, ability to fly, and survival after emergence are relevant for the successful continuity of a population, 200 mg kg-1 sediment dry weight (nominal) is suggested as the overall NOEC to be derived from this study. This is 102 mg/kg dw expressed as the actually measured concentration.

Validity criteria fulfilled:
yes
Remarks:
pH, oxygen saturation and temperature within range and stable, emergence fast enough and mean emergence above 70%
Conclusions:
The 28d-NOEC of OTNE towards Chironmus riparius based on additional observations: complete emergence, ability to fly and survival after emergence is 200 mg/kg dw (nominal). The 28d-NOEC expressed on the basis of the actual concentration is 102 mg/kg dw ((with total carbon content 2.24%).
Executive summary:

The chronic toxicity of OTNE towards Chironomus riparius in sediment was investigated according to methodology comparable to OECD Guideline 218 under GLP. Validity criteria for the test were met. Organisms were exposed to nominal concentrations of 50, 100, 200, 400 and 1000 mg/kg dw of test material in the sediment. The actual concentrations in the sediment on day 0 were 50 to 67% of the target concentrations. At termination of the test the concentration was 84-89% of the concentration on day 0 (Belfroid and Blok 2006). The emergence ratio was not affected up to 400 mg/kg, whereas the development rate was not affected in any of the test concentrations. At concentrations ≥ 400 mg/kg(d.w.) an increasing percentage of fully emerged midges were dead and/or not able to fly (ratite) and also the percentage of dead, incompletely emerged midges increased at 400 - 1000 mg/kg (Egeler and Gilberg 2005a).Therefore the NOEC was 200 mg/kg dw (nominal); after correction for the concentrations on day 0, the actual NOEC was 102 mg/kg dw (Belfroid and Blok 2006).

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15/04/2005 - 31/10/2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to methodology comparable to OECD TG 218 in compliance with GLP, without deviations that influence the quality of the results. Includes analysis of actual concentrations.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)
Deviations:
yes
Remarks:
Non-Chironomid-species was used. Instead of emergence and development, surivival, amphipod length and total biomass were used to determine toxicity values. The analytical investigations were outsourced and reported separately (Belfroid&Blok, 2006)
Qualifier:
equivalent or similar to
Guideline:
other: U.S. EPA (2000): "Methods for measuring the toxicity and bioaccumulation of sediment-associated contaminants with freshwater invertebrates. Second Edition. EPA 600/R-99/064, U.S. Environmental Protection Agency, Duluth, MN, March 2000".
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
SAMPLING PROCEDURE
Samples of sediment, pore water and overlying water were taken during the exposure up to 28 days. The overlying water was taken directly from the test vessels by carefully decanting into a glass beaker. The wet sediment was centrifuged at 9425 x g. The pore water was carefully decanted into glass beakers. All samples were deep-frozen at -18°C to -25°C.
- Sampling interval: directly after spiking (day -7), at the end of equilibration (day 0), on day 14 of exposure and at the end of the exposure period (day 28)

SEDIMENT
- Concentrations: 1.2, 4.0, 12.7, 40.6 and 130 mg/kg sediment dry weight (nominal)
- Sample handling: Sediment samples were defrosted, homogenized and a subsample was taken for dry weight determination as described by Egeler et al (2005a). The remaining subsample was freeze-dried. With every run of freeze-drying, a blank sample consisting of 1 g of silicagel was included. A subsample of 2 – 3 g of dried material was collected for shipment. The recovery standard D3-AHTN was added to both freeze dried sediment and blank silicagel.
- Sample storage before analysis: deep-frozen at -18 to -25 degrees Celsius

PORE WATER
- Sample Handling: Frozen samples of overlying water and pore water were defrosted at room temperature. The recovery standard D3-AHTN (deuterated AHTN) was added and the samples were extracted with Speedisks as described by Egeler et al (2005a) and frozen again for shipment
- Sample storage before analysis: deep-frozen at -18 to -25 degrees Celsius

OVERLYING WATER
- Concentrations: 41, 131, 419, 1342 and 4295 mg/l (nominal)
- Sample handling and storage as for pore water
Vehicle:
yes
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: Artificial sediment was formulated. The sediment was amended prior to the application of the test item with finely ground leaves of stinging nettle (Urtica sp.; urtica powder) and cellulose (α-cellulose powder) to an amount of 0.4 - 0.5% of sediment dry weight to ensure that the animals will reproduce under control conditions. In this way it is ensured that the test animals were exposed through the water and sediment as well as by their food.
- Method of mixing: no data
- Details of spiking: The test solutions for application of test item into the sediment were obtained by diluting the stock solution with acetone. From these test solutions an appropriate volume was mixed with an appropriate amount of quartz sand for each treatment (10 g of quartz sand per replicate). The acetone was evaporated completely before the sediment of each concentration level was prepared. The amount of sand provided by the test-item-and-sand mixture was taken into account when preparing the formulated sediment. To ensure that the test item added to sediment was evenly distributed within the sediment, the bulk formulated sediments were thoroughly mixed. From these bulk concentration levels, the sediment was distributed to the individual replicates of each concentration level.
- Equilibration time: 7 days
- Equilibration conditions: same as during test
- Controls: yes, blanks and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 100% in stock solution,
- Evaporation of vehicle before use:
Test organisms (species):
Hyalella azteca
Details on test organisms:
The test organism Hyalella azteca (Saussure), a sediment-dwelling freshwater amphipod, has been cultured at ECT Oekotoxikologie GmbH since
October 2002. The animals were originally obtained from Dresden University of Technology.
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
Post exposure observation period:
In order to estimate mortalities, the number of amphipods that did not react to a gentle stimulus, and the number of missing amphipods were considered dead.
During the exposure, observations concerning the behaviour did not reveal any concentration-dependent differences between treatments and control vessels.
Hardness:
367.9 - 417.9 mg CaCO3 /L (20.6 - 23.4 °dH)
Test temperature:
19.8 - 20.4 oC
pH:
7.9 - 8.6
Dissolved oxygen:
7.5 - 9.2 mg/L
Ammonia:
0.2 - 2.7 mg NH4+ /L
Nominal and measured concentrations:
Sediment (nominal): 1.2, 4.0, 12.7, 40.6 and 130 mg/kg sediment dry weight
Overlying water (nominal): 41, 131, 419, 1342 and 4295 mg/l (nominal)
Actual concentrations were measured in the middle (C3) and highest (C5) concentrations, in sediment, overlying water and porewater on days 0, 14 and 28. The results of the analyses showed that the concentrations of OTNE decreased gradually (from day -7 to day 0), but stayed above 80% of theconcentration at the start of the exposure (between day 0 and day 28). The level of the NOEC (40.6 mg/kg, C4) was not included in the samples, but the actual concentration was derived based on the average decrease in the concentrations in C3 and C5. Thus the NOEC was 18.4 mg/kg dw (actual concentration).
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
18.4 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
and total biomass
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
40.6 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: total biomass
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
40.6 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 130 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: amphipod length
Duration:
28 d
Dose descriptor:
other: EC15
Effect conc.:
61 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: total biomass
Duration:
28 d
Dose descriptor:
other: EC15
Effect conc.:
77.4 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
197.9 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
218.5 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: total biomass
Details on results:
Mortality observed ranged from 1.52 % at the lowest concentration (1.2 mg/kg dw) to 34.34 % at the highest concentration of 130 mg/kg dw.
During exposure, observations concerning behaviour did not reveal any concentration-dependent differences between treatments and control vessels.
Validity criteria fulfilled:
yes
Remarks:
pH, dissolved oxygen, temperature within range and stable throughout the test period. Mean mortality in control vessels 0%.
Conclusions:
The 28d-NOEC of OTNE towards the reproduction and mortality of Hyalella azteca are (nominal) 40.6 mg/kg sediment dry weight. The 28d-NOEC expressed on the basis of the actual concentration was 18.4 mg/kg dw (with total organic carbon content of 2.33%).
Executive summary:

The chronic toxicity of OTNE towards Hylella azteca was investigated according to methodology comparable to OECD Guideline 208, under GLP. The actual concentrations on day 0 of the test with Hyalella azteca were 43 to 47% of the intended concentrations in the sediment. On day 28 the concentrations were within 80% of the concentrations on day 0 (Belfroid and Blok 2005). Survival and biomass were not affected up to 40.6 mg/kg dw (NOEC), whereas growth was not affected up to the highest concentration of 130 mg/kg dw (Egeler and Gilberg 2005b). After correction for the concentrations on day 0 the actual NOEC was 18.4 mg/kg dw (Belfroid and Blok 2006).  

Description of key information

For three different sediment species representing different living and feeding conditions, the NOEC in a 28d-test is available. The lowest NOEC, based on measured concentrations, is  17.1 mg/kg dw (Total Organic Carbon 2.20%). 

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:
17.1 mg/kg sediment dw

Additional information

Three invertebrate species associated with the sediment and representing different living and feeding conditions, were tested according to or in line with OECD TG 218 in compliance with GLP. The results of these tests are summarised in the below table. More information on the tests is provided below the table.

Test species

Guideline

Results (mg/kg dw) and 95% confidence limits

Remarks

Lumbriculus variegatus

 

Comparable to OECD 218

28d-NOECreprod.& 28d-NOECbiomass= 33.3 mg/kg

28d-EC50reprod. = 97 mg/kg.

Egeler and Gilberg 2005c

Total organic carbon 2.29%

nom. 33.3 mg/kg

~measured 17.1 mg/kg

Hyalella azteca

 

Comparable to OECD 218

28d-NOECsurvival& 28d-NOECbiomass= 40.6 mg/kg

28d-EC50survival= 198 <115 – 339> mg/kg

Egeler and Gilberg 2005b

Total organic carbon 2.33%

nom. 40.6 mg/kg,

~measured 18.4 mg/kg

 

Chironomus riparius

 

Comparable to OECD 218

28d-NOECemergence ratio= 400 mg/kg

28d-NOECratite midges= 200 mg/kg

28d-EC50emergence ratio= 642 <516-797> mg/kg

Egeler and Gilberg 2005a

Total organic carbon 2.24%

nom. 200 mg/kg,

~measured 102 mg/kg

 

The resulting NOECs are expressed based on the measured concentrations as indicated in the third column of the above table. For the safety assessment, the results are to be converted to a sediment containing 5% TOC in order to be comparable to the estimations for a 'standard sediment'.

The sediment was formulated from quartz sand, kaolinite clay and Sphagnum moss peat. Urtica powder was added as feed. The total organic carbon content was 2%. The sediment including the feed was spiked with test material solved in acetone. Each vessel contained a layer of sediment and the water volume was circa 4 times the sediment volume. The test animals were introduced after an equilibrium period of 1 week. Test concentrations were measured in the sediment, porewater as well as in overlying water.

 

In the test with Lumbriculus variegatus, the test organisms were exposed to nominal concentrations of 1.2, 3.7, 11.1, 33.3 and 100 mg/kg sediment dry weight. The actual concentrations in the sediment upon introduction of the test organisms were 52% of nominal. On day 14 the measured concentrations were within 80% of the concentrations on day 0. This was also the case for the highest concentration on day 28, but middle concentration fell below 80% (Belfroid and Blok 2006). Survival was not affected up to the highest test concentration of 100 mg/kg dw. Reproduction and growth were not affected up to 33 mg/kg dw. The reproduction was inhibited by 55% and growth was inhibited by 35% at 100 mg/kg (Egeler and Gilberg 2005). After correction for the actual concentration on day 0, the actual NOEC was 17.1 mg/kg dw (Belfroid and Blok 2006).

In the test with Chironomus riparius, the test organisms were exposed to nominal concentrations of 50, 100, 200, 400 and 1000 mg/kg dw of test material in the sediment. The actual concentrations in the sediment on day 0 were 50 to 67% of the target concentrations. At termination of the test the concentration was 84-89% of the concentration on day 0 (Belfroid and Blok 2006). The emergence ratio was not affected up to 400 mg/kg, whereas the development rate was not affected in any of the test concentrations. At concentrations ≥ 400 mg/kg(d.w.) an increasing percentage of fully emerged midges were dead and/or not able to fly (ratite) and also the percentage of dead, incompletely emerged midges increased at 400 - 1000 mg/kg (Egeler and Gilberg 2005a).Therefore the NOEC was 200 mg/kg dw (nominal); after correction for the concentrations on day 0, the actual NOEC was 102 mg/kg dw (Belfroid and Blok 2006).

In the test with Hyalella azteca, the test organisms were exposed to nominal concentrations of 1.2, 4.0, 12.7, 40.6 and 130 mg/kg sediment dry weight. The actual concentrations on day 0 of the test with Hyalella azteca were 43 to 47% of the intended concentrations in the sediment. On day 28 the concentrations were within 80% of the concentrations on day 0 (Belfroid and Blok 2005). Survival and biomass were not affected up to 40.6 mg/kg dw (NOEC), whereas growth was not affected up to the highest concentration of 130 mg/kg dw (Egeler and Gilberg 2005b). After correction for the concentrations on day 0 the actual NOEC was 18.4 mg/kg dw (Belfroid and Blok 2006).