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Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 September 2018 to December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ISO standard 10872
Version / remarks:
2010 / Water quality -- Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda)
GLP compliance:
no
Remarks:
Testing house has longterm experience with the nematode test and participated in ring tests within the standardization process of ISO 10872
Specific details on test material used for the study:
SSA 415 m2/g, water solubility 232.6 mg/L
Analytical monitoring:
no
Details on sampling:
6 replicates/treatment group;
10 test organisms/replicate
Vehicle:
no
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Artifical sediment according to ISO standard was used (41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content);
- Details of spiking: artifical sediment was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). The test substance was directly added to sediment.
- Equilibration time: 24 hours
- Equilibration conditions: 6 °C
- Controls: sediment, non-spiked
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none used
Test organisms (species):
nematodes
Details on test organisms:
Caenorhabditis elegans, strain N2; genotype: C. elegans wild type, DR subclone of CB original (Tc1 pattern);
Source: Caenorhabditis Genetic Centre; University of Minnesota, Minneapolis, USA;
first juvenile stage (J1) at start of the test (initial body length: Test 1: 315 (± 34) µm; Test 2: 332 (± 52) µm)
Cultivation of test organisms: according to ISO 10872
Study type:
laboratory study
Test type:
static
Type of sediment:
artificial sediment
Limit test:
no
Duration:
96 h
Test temperature:
19.9-20.4°C
Nominal and measured concentrations:
0 (control), 0.1, 0.5, 1, 5, 10g/kg dry sediment (nominal), corresponding to 0, 0.06, 0.3, 0.6, 3 and 6 g/kg wet weight (nominal)
Details on test conditions:
TEST SYSTEM
- Test container: 12-well multidishes
- Sediment volume: 0.300 (± 0.003) g of the dry spiked sediment or the controls were weighed into each test well
- Weight of wet sediment with and without water: 0.2 ml M9-medium (ISO 10872; 6 g Na2HPO4/L, 3 g KH2PO4/L, 5 g NaCl, 0.25 g MgSO4 7H2O/L) were added to 0.3 g dry sediment to moisten the sediment and to achieve a sediment wet weight of 0.5g and a water content of 40%

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 6
- Feeding: Immediately before the start of the test, 0.5 mL of bacterial suspension (Escherichia coli OP50 n M9-medium; cell density: 12,000 ± 600 FAU; according to ISO 10872) were added to the 0.5 g wet sediment and thoroughly mixed with a spatula, achieving 1 g of wet sediment with 0.300 (± 0.003) g dry weight.)

After 96 hours, all wells were stained with Bengal rose and heat treated at 80 °C for 10 minutes; afterwards stored at 8 °C (in accordance with ISO 10872)

EXTRACTION OF NEMATODES FROM TESTED SEDIMENTS
After termination of the test, test organisms and offspring are separated from the sediment by a flotation technique using Ludox TM50 (50 wt % suspension in water; Sigma-Aldrich; Batch: 07826PH; adjusted with water to a density of 1.13 g/mL). After mixing the sediment with Ludox-suspension and subsequent centrifugation, nematodes are found in the supernatant. After three extraction steps, the combined supernatant is analyzed. The added Ludox TM50 suspension has no impact on the test results, as it is added after the test has been terminated.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
10 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
5 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
10 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Details on results:
For nematode growth, first significant inhibitory effects occurred at 10 g kg-1 dry sediment (6 g kg-1 wet sediment) (p < 0.05; one-way ANOVA; post-hoc Dunnett). Thus, for growth a LOEC and NOEC of 10 and 5 g kg-1 dry sediment (6 and 3 g kg-1 wet sediment) could be identified, respectively. Reproduction was not affected up to the highest tested concentration of 10 g kg-1 dry sediment. Therefore, for reproduction a LOEC could not be identified.
Results with reference substance (positive control):
no reference substance used
Reported statistics and error estimates:
one-way ANOVA (post-hoc test: Dunnett’s test), descriptive statistics

Table: Validity criteria in controls; x = fulfilled

Validity Criteria

Test 1

Test 2

% Recovery in Controls (80 ≤ x ≤ 120%)

91.7 (x)

90.0 (x)

% Males in Controls

(≤ 10%)

0.0 (x)

0.0 (x)

% Fertility in Controls

(≥ 80%)

99.1 (x)

100.0 (x)

Reproduction in Controls (≥ 30 offspring / test organisms)

119 (x)

124 (x)

 

Validity criteria fulfilled:
yes
Conclusions:
A decrease in body length was found at 10 g/kg sediment (dw); no effect was found on reproduction up to the highest tested concentration of 10 g/kg sediment (dw). Therefore a nominal NOEC of 5 g/kg sediment (dw) was derived from this study.
Executive summary:

During the study artificial sediment (with particle size distribution of 41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). For spiking of sediment the test substance was directly added to sediment. In order to allow the test substance to equally distribute between aqueous and solid phase, the spiked sediment was incubated for 24 h before the start of the test.

Test parameters were growth (body length in µm), and reproduction (number of offspring divided by number of introduced test organisms). Before the start of the assay, 0.5 mL of bacterial suspension (E. coli in M9 medium) were added to each vessel as food for the nematodes. After that 10 juvenile worms of the first stage (J1) were added to each vessel, containing now 0.5 g sediment (ww), 0.5 mL test solution and 0.5 mL bacterial suspension. The vessels were then incubated for 96 h. Six replicates per treatment group were set up for the test. In order to stop the assay, the nematodes were heat killed and stained with Rose Bengal. After extracting the nematodes from the sediment, body length and number of offspring were determined. A decrease in body length was found at 10 g/kg sediment (dw); no effect was found on reproduction up to the highest tested concentration of 10 g/kg sediment (dw). Therefore a nominal NOEC of 5 g/kg sediment (dw) was derived from this study.

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 November 2018 to 11 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ISO standard 10872
Version / remarks:
2010 / Water quality -- Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda)
GLP compliance:
no
Remarks:
Testing house has longterm experience with the nematode test and participated in ring tests within the standardization process of ISO 10872
Specific details on test material used for the study:
particle size 15µm; "primary particle" size 7nm, specific surface area 250m2/g, water solubility 110mg/L
Analytical monitoring:
no
Details on sampling:
6 replicates/treatment group; 12 replicates in the control group;
10 test organisms/replicate
Vehicle:
no
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Artifical sediment according to ISO standard was used (41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content);
- The artifical sediment was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). The test substance was directly added to sediment.
- Equilibration time: 24 hours
- Equilibration conditions: 6 °C
- Controls: sediment, non-spiked
Test organisms (species):
nematodes
Details on test organisms:
Caenorhabditis elegans, strain N2; genotype: C. elegans wild type, DR subclone of CB original (Tc1 pattern);
Source: Caenorhabditis Genetic Centre; University of Minnesota, Minneapolis, USA;
first juvenile stage (J1) at start of the test (initial body length: Test 1: 315 (± 34) µm; Test 2: 332 (± 52) µm)
Cultivation of test organisms: according to ISO 10872
Study type:
laboratory study
Test type:
static
Type of sediment:
artificial sediment
Limit test:
no
Duration:
96 h
Test temperature:
19.8 – 20.3 °C
Nominal and measured concentrations:
0 (control), 0.1, 0.5, 1, 5, 10g/kg dry sediment (nominal), corresponding to 0, 0.06, 0.3, 0.6, 3 and 6 g/kg wet weight (nominal)
Details on test conditions:
TEST SYSTEM
- Test container: 12-well multidishes
- Sediment volume: 0 .300 (± 0.003) g of the dry spiked sediment or the controls were weighed into each test well
- Weight of wet sediment with and without water: 0.2 ml M9-medium (ISO 10872; 6 g Na2HPO4/L, 3 g KH2PO4/L, 5 g NaCl, 0.25 g MgSO4 7H2O/L) were added to 0.3 g dry sediment to moisten the sediment and to achieve a sediment wet weight of 0.5g and a water content of 40%

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 12
- Feeding: Immediately before the start of the test, 0.5 mL of bacterial suspension (Escherichia coli OP50 n M9-medium; cell density: 12,000 ± 600 FAU; according to ISO 10872) were added to the 0.5 g wet sediment and thoroughly mixed with a spatula, achieving 1 g of wet sediment with 0.300 (± 0.003) g dry weight.)

After 96 hours, all wells were stained with Bengal rose and heat treated at 80 °C for 10 minutes; afterwards stored at 8 °C (in accordance with ISO 10872)

EXTRACTION OF NEMATODES FROM TESTED SEDIMENTS
After termination of the test, test organisms and offspring are separated from the sediment by a flotation technique using Ludox TM50 (50 wt % suspension in water; Sigma-Aldrich; Batch: 07826PH; adjusted with water to a density of 1.13 g/mL). After mixing the sediment with Ludox-suspension and subsequent centrifugation, nematodes are found in the supernatant. After three extraction steps, the combined supernatant is analyzed. The added Ludox TM50 suspension has no impact on the test results, as it is added after the test has been terminated.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
5 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
10 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
5 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
8.5 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
5.4 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Details on results:
Significant effects on nematode growth occurred at 5 g/kg dry sediment (p < 0.05; one-way ANOVA; post-hoc Dunnett test; LOEC). The NOEC was at 1 g/kg dry sediment. Inhiibitory effects on reproduction were found at 10 g/kg dry sediment (p<0.05; one-way ANOVA; posthoc Games-Howell: LOEC). The NOEC was at 5 g/kg dry sediment.
Results with reference substance (positive control):
no reference substance used
Reported statistics and error estimates:
one-way ANOVA (post-hoc test: Dunnett’s test), descriptive statistics

Table: Validity criteria in controls; x = fulfilled

Validity Criteria

Test

 

% Recovery in Controls (80 ≤ x ≤ 120%)

91.7 (x)

 

% Males in Controls

(≤ 10%)

0.0 (x)

 

% Fertility in Controls

(≥ 80%)

100 (x)

 

Reproduction in Controls (≥ 30 offspring / test organisms)

136 (x

 

Validity criteria fulfilled:
yes
Conclusions:
A decrease in body length was found at 5 g/kg sediment (dw); an effect on reproduction was found at 10 g/kg sediment (dw). A NOEC of 1 g/kg sediment (dw) was derived from this study.
Executive summary:

During the study artificial sediment (with particle size distribution of 41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). For spiking of sediment the test substance was directly added to sediment. In order to allow the test substance to equally distribute between aqueous and solid phase, the spiked sediment was incubated for 24 h before the start of the test.

Test parameters were growth (body length in µm), and reproduction (number of offspring divided by number of introduced test organisms). Before the start of the assay, 0.5 mL of bacterial suspension (E. coli in M9 medium) were added to each vessel as food for the nematodes. After that 10 juvenile worms of the first stage (J1) were added to each vessel, containing now 0.5 g sediment (ww), 0.5 mL test solution and 0.5 mL bacterial suspension. The vessels were then incubated for 96 h. Six replicates per treatment group were set up for the test. In order to stop the assay, the nematodes were heat killed and stained with Rose Bengal. After extracting the nematodes from the sediment, body length and number of offspring were determined. A decrease in body length was found at 5 g/kg sediment (dw); an effect on reproduction was found at 10 g/kg sediment (dw). A NOEC of 1 g/kg sediment (dw) was derived from this study.

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 November 2018 to 11 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ISO standard 10872
Version / remarks:
2010 / Water quality -- Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda)
GLP compliance:
no
Remarks:
Testing house has longterm experience with the nematode test and participated in ring tests within the standardization process of ISO 10872
Specific details on test material used for the study:
particle size 15µm; "primary particle" size 7nm, specific surface area 250m2/g, water solubility 110mg/L
Analytical monitoring:
no
Details on sampling:
6 replicates/treatment group; 12 replicates in the control group;
10 test organisms/replicate
Vehicle:
no
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Artifical sediment according to ISO standard was used (41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content);
- Details of spiking: artifical sediment was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). The test substance was directly added to sediment.
- Equilibration time: 24 hours
- Equilibration conditions: 6 °C
- Controls: sediment, non-spiked
Test organisms (species):
nematodes
Details on test organisms:
Caenorhabditis elegans, strain N2; genotype: C. elegans wild type, DR subclone of CB original (Tc1 pattern);
Source: Caenorhabditis Genetic Centre; University of Minnesota, Minneapolis, USA;
first juvenile stage (J1) at start of the test (initial body length: Test 1: 315 (± 34) µm; Test 2: 332 (± 52) µm)
Cultivation of test organisms: according to ISO 10872
Study type:
laboratory study
Test type:
static
Type of sediment:
artificial sediment
Limit test:
no
Duration:
96 h
Test temperature:
19.8 – 20.3 °C
Nominal and measured concentrations:
0 (control), 0.1, 0.5, 1, 5, 10g/kg dry sediment (nominal), corresponding to 0, 0.06, 0.3, 0.6, 3 and 6 g/kg wet weight (nominal)
Details on test conditions:
TEST SYSTEM
- Test container: 12-well multidishes
- Sediment volume: 0 .300 (± 0.003) g of the dry spiked sediment or the controls were weighed into each test well
- Weight of wet sediment with and without water: 0.2 ml M9-medium (ISO 10872; 6 g Na2HPO4/L, 3 g KH2PO4/L, 5 g NaCl, 0.25 g MgSO4 7H2O/L) were added to 0.3 g dry sediment to moisten the sediment and to achieve a sediment wet weight of 0.5g and a water content of 40%

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 12
- Feeding: Immediately before the start of the test, 0.5 mL of bacterial suspension (Escherichia coli OP50 n M9-medium; cell density: 12,000 ± 600 FAU; according to ISO 10872) were added to the 0.5 g wet sediment and thoroughly mixed with a spatula, achieving 1 g of wet sediment with 0.300 (± 0.003) g dry weight.)

After 96 hours, all wells were stained with Bengal rose and heat treated at 80 °C for 10 minutes; afterwards stored at 8 °C (in accordance with ISO 10872)

EXTRACTION OF NEMATODES FROM TESTED SEDIMENTS
After termination of the test, test organisms and offspring are separated from the sediment by a flotation technique using Ludox TM50 (50 wt % suspension in water; Sigma-Aldrich; Batch: 07826PH; adjusted with water to a density of 1.13 g/mL). After mixing the sediment with Ludox-suspension and subsequent centrifugation, nematodes are found in the supernatant. After three extraction steps, the combined supernatant is analyzed. The added Ludox TM50 suspension has no impact on the test results, as it is added after the test has been terminated.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
5 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
10 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
5 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
8.5 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
5.4 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Details on results:
Significant effects on nematode growth occurred at 5 g/kg dry sediment (p < 0.05; one-way ANOVA; post-hoc Dunnett test; LOEC). The NOEC was at 1 g/kg dry sediment. Inhiibitory effects on reproduction were found at 10 g/kg dry sediment (p<0.05; one-way ANOVA; posthoc Games-Howell: LOEC). The NOEC was at 5 g/kg dry sediment.
Results with reference substance (positive control):
no reference substance used
Reported statistics and error estimates:
one-way ANOVA (post-hoc test: Dunnett’s test), descriptive statistics

Table: Validity criteria in controls; x = fulfilled

Validity Criteria

Test

 

% Recovery in Controls (80 ≤ x ≤ 120%)

91.7 (x)

 

% Males in Controls

(≤ 10%)

0.0 (x)

 

% Fertility in Controls

(≥ 80%)

100 (x)

 

Reproduction in Controls (≥ 30 offspring / test organisms)

136 (x

 

Validity criteria fulfilled:
yes
Conclusions:
A decrease in body length was found at 5 g/kg sediment (dw); an effect on reproduction was found at 10 g/kg sediment (dw). A NOEC of 1 g/kg sediment (dw) was derived from this study.
Executive summary:

During the study artificial sediment (with particle size distribution of 41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). For spiking of sediment the test substance was directly added to sediment. In order to allow the test substance to equally distribute between aqueous and solid phase, the spiked sediment was incubated for 24 h before the start of the test.

Test parameters were growth (body length in µm), and reproduction (number of offspring divided by number of introduced test organisms). Before the start of the assay, 0.5 mL of bacterial suspension (E. coli in M9 medium) were added to each vessel as food for the nematodes. After that 10 juvenile worms of the first stage (J1) were added to each vessel, containing now 0.5 g sediment (ww), 0.5 mL test solution and 0.5 mL bacterial suspension. The vessels were then incubated for 96 h. Six replicates per treatment group were set up for the test. In order to stop the assay, the nematodes were heat killed and stained with Rose Bengal. After extracting the nematodes from the sediment, body length and number of offspring were determined. A decrease in body length was found at 5 g/kg sediment (dw); an effect on reproduction was found at 10 g/kg sediment (dw). A NOEC of 1 g/kg sediment (dw) was derived from this study.

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 September 2018 to December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ISO standard 10872
Version / remarks:
2010 / Water quality -- Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda)
GLP compliance:
no
Remarks:
Testing house has longterm experience with the nematode test and participated in ring tests within the standardization process of ISO 10872
Specific details on test material used for the study:
remains in suspension as monodisperse particle, particle size 7nm, specific surface area 130-180m2/g, water solubility 122mg/L
Analytical monitoring:
no
Details on sampling:
6 replicates/treatment group; 12 replicates in the control group;
10 test organisms/replicate
Vehicle:
no
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Artifical sediment according to ISO standard was used (41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content);
- Details of spiking: artifical sediment was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). The test substance was directly added to sediment.
- Equilibration time: 24 hours
- Equilibration conditions: 6 °C
- Controls: sediment, non-spiked
Test organisms (species):
nematodes
Details on test organisms:
Caenorhabditis elegans, strain N2; genotype: C. elegans wild type, DR subclone of CB original (Tc1 pattern);
Source: Caenorhabditis Genetic Centre; University of Minnesota, Minneapolis, USA;
first juvenile stage (J1) at start of the test (initial body length: Test 1: 315 (± 34) µm; Test 2: 332 (± 52) µm)
Cultivation of test organisms: according to ISO 10872
Study type:
laboratory study
Test type:
static
Type of sediment:
artificial sediment
Limit test:
no
Duration:
96 h
Test temperature:
19.9 – 20.4 °C
Nominal and measured concentrations:
0 (control), 0.1, 0.5, 1, 5, 10g/kg dry sediment (nominal), corresponding to 0, 0.06, 0.3, 0.6, 3 and 6 g/kg wet weight (nominal)
Details on test conditions:
TEST SYSTEM
- Test container: 12-well multidishes
- Sediment volume: 0 .300 (± 0.003) g of the dry spiked sediment or the controls were weighed into each test well
- Weight of wet sediment with and without water: 0.2 ml M9-medium (ISO 10872; 6 g Na2HPO4/L, 3 g KH2PO4/L, 5 g NaCl, 0.25 g MgSO4 7H2O/L) were added to 0.3 g dry sediment to moisten the sediment and to achieve a sediment wet weight of 0.5g and a water content of 40%

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 12
- Feeding: Immediately before the start of the test, 0.5 mL of bacterial suspension (Escherichia coli OP50 n M9-medium; cell density: 12,000 ± 600 FAU; according to ISO 10872) were added to the 0.5 g wet sediment and thoroughly mixed with a spatula, achieving 1 g of wet sediment with 0.300 (± 0.003) g dry weight.)

After 96 hours, all wells were stained with Bengal rose and heat treated at 80 °C for 10 minutes; afterwards stored at 8 °C (in accordance with ISO 10872)

EXTRACTION OF NEMATODES FROM TESTED SEDIMENTS
After termination of the test, test organisms and offspring are separated from the sediment by a flotation technique using Ludox TM50 (50 wt % suspension in water; Sigma-Aldrich; Batch: 07826PH; adjusted with water to a density of 1.13 g/mL). After mixing the sediment with Ludox-suspension and subsequent centrifugation, nematodes are found in the supernatant. After three extraction steps, the combined supernatant is analyzed. The added Ludox TM50 suspension has no impact on the test results, as it is added after the test has been terminated.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
10 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
5 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
10 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Details on results:
Significant effects on nematode reproduction occurred at 10 g/kg dry sediment (p < 0.05; one-way ANOVA; post-hoc Dunnett test; LOEC). The NOEC was at 5 g/kg dry sediment. No effects were noted on growth.
Results with reference substance (positive control):
no reference substance used
Reported statistics and error estimates:
one-way ANOVA (post-hoc test: Dunnett’s test), descriptive statistics

Table:Validity criteria in controls; x = fulfilled

Validity Criteria

Test 1

Test 2

% Recovery in Controls (80 ≤ x ≤ 120%)

91.7 (x)

90.0 (x)

% Males in Controls

(≤ 10%)

0.0 (x)

0.0 (x)

% Fertility in Controls

(≥ 80%)

99.1 (x)

100.0 (x)

Reproduction in Controls (≥ 30 offspring / test organisms)

119 (x)

124 (x)

 

Validity criteria fulfilled:
yes
Conclusions:
A decrease in reproduction was found at 10 g/kg sediment (dw); no effect were found on growth.
Executive summary:

During the study artificial sediment (with particle size distribution of 41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). For spiking of sediment the test substance was directly added to sediment. In order to allow the test substance to equally distribute between aqueous and solid phase, the spiked sediment was incubated for 24 h before the start of the test.

Test parameters were growth (body length in µm), and reproduction (number of offspring divided by number of introduced test organisms). Before the start of the assay, 0.5 mL of bacterial suspension (E. coli in M9 medium) were added to each vessel as food for the nematodes. After that 10 juvenile worms of the first stage (J1) were added to each vessel, containing now 0.5 g sediment (ww), 0.5 mL test solution and 0.5 mL bacterial suspension. The vessels were then incubated for 96 h. Six replicates per treatment group were set up for the test. In order to stop the assay, the nematodes were heat killed and stained with Rose Bengal. After extracting the nematodes from the sediment, body length and number of offspring were determined. A decrease in reproduction was found at 10 g/kg sediment (dw); an effect on growth was not found. A NOEC of 5 g/kg sediment (dw) was derived from this study.

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 September 2018 to December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ISO standard 10872
Version / remarks:
2010 / Water quality -- Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda)
GLP compliance:
no
Remarks:
Testing house has longterm experience with the nematode test and participated in ring tests within the standardization process of ISO 10872
Specific details on test material used for the study:
precipitated silica, particle size 6.3-7.2 µm, "primary particle" size <20 nm, surface area 752m2/g, water solubility 133 mg/L
Analytical monitoring:
no
Details on sampling:
6 replicates/treatment group;
10 test organisms/replicate
Vehicle:
no
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Artifical sediment according to ISO standard was used (41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content);
- Details of spiking: artifical sediment was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). The test substance was directly added to sediment.
- Equilibration time: 24 hours
- Equilibration conditions: 6 °C
- Controls: sediment, non-spiked
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none used
Test organisms (species):
nematodes
Details on test organisms:
Caenorhabditis elegans, strain N2; genotype: C. elegans wild type, DR subclone of CB original (Tc1 pattern);
Source: Caenorhabditis Genetic Centre; University of Minnesota, Minneapolis, USA;
first juvenile stage (J1) at start of the test (initial body length: Test 1: 315 (± 34) µm; Test 2: 332 (± 52) µm)
Cultivation of test organisms: according to ISO 10872
Study type:
laboratory study
Test type:
static
Type of sediment:
artificial sediment
Limit test:
no
Duration:
96 h
Test temperature:
19.9-20.4°C
Nominal and measured concentrations:
0 (control), 0.1, 0.5, 1, 5, 10g/kg dry sediment (nominal), corresponding to 0, 0.06, 0.3, 0.6, 3 and 6 g/kg wet weight (nominal)
Details on test conditions:
TEST SYSTEM
- Test container: 12-well multidishes
- Sediment volume: 0.300 (± 0.003) g of the dry spiked sediment or the controls were weighed into each test well
- Weight of wet sediment with and without water: 0.2 ml M9-medium (ISO 10872; 6 g Na2HPO4/L, 3 g KH2PO4/L, 5 g NaCl, 0.25 g MgSO4 7H2O/L) were added to 0.3 g dry sediment to moisten the sediment and to achieve a sediment wet weight of 0.5g and a water content of 40%

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 6
- Feeding: Immediately before the start of the test, 0.5 mL of bacterial suspension (Escherichia coli OP50 n M9-medium; cell density: 12,000 ± 600 FAU; according to ISO 10872) were added to the 0.5 g wet sediment and thoroughly mixed with a spatula, achieving 1 g of wet sediment with 0.300 (± 0.003) g dry weight.)

After 96 hours, all wells were stained with Bengal rose and heat treated at 80 °C for 10 minutes; afterwards stored at 8 °C (in accordance with ISO 10872)

EXTRACTION OF NEMATODES FROM TESTED SEDIMENTS
After termination of the test, test organisms and offspring are separated from the sediment by a flotation technique using Ludox TM50 (50 wt % suspension in water; Sigma-Aldrich; Batch: 07826PH; adjusted with water to a density of 1.13 g/mL). After mixing the sediment with Ludox-suspension and subsequent centrifugation, nematodes are found in the supernatant. After three extraction steps, the combined supernatant is analyzed. The added Ludox TM50 suspension has no impact on the test results, as it is added after the test has been terminated.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
10 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
10 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Details on results:
For nematode growth, first significant inhibitory effects occurred at 10 g kg-1 dry sediment (6 g kg-1 wet sediment) (p < 0.05; one-way ANOVA; post-hoc Dunnett). Thus, for growth a LOEC and NOEC of 10 and 5 g kg-1 dry sediment (6 and 3 g kg-1 wet sediment) could be identified, respectively. Reproduction was not affected up to the highest tested concentration of 10 g kg-1 dry sediment. Therefore, for reproduction a LOEC could not be identified.
Results with reference substance (positive control):
no reference substance used
Reported statistics and error estimates:
one-way ANOVA (post-hoc test: Dunnett’s test), descriptive statistics

Table: Validity criteria in controls; x = fulfilled

Validity Criteria

Test 1

Test 2

% Recovery in Controls (80 ≤ x ≤ 120%)

91.7 (x)

90.0 (x)

% Males in Controls

(≤ 10%)

0.0 (x)

0.0 (x)

% Fertility in Controls

(≥ 80%)

99.1 (x)

100.0 (x)

Reproduction in Controls (≥ 30 offspring / test organisms)

119 (x)

124 (x)

 

Validity criteria fulfilled:
yes
Conclusions:
A decrease in body length was found at 10 g/kg sediment (dw); no effect was found on reproduction up to and at the highest tested concentration of 10 g/kg sediment (dw). A NOEC of 5 g/kg sediment (dw) was derived from this study.
Executive summary:

During the study artificial sediment (with particle size distribution of 41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). For spiking of sediment the test substance was directly added to sediment. In order to allow the test substance to equally distribute between aqueous and solid phase, the spiked sediment was incubated for 24 h before the start of the test.

Test parameters were growth (body length in µm), and reproduction (number of offspring divided by number of introduced test organisms). Before the start of the assay, 0.5 mL of bacterial suspension (E. coli in M9 medium) were added to each vessel as food for the nematodes. After that 10 juvenile worms of the first stage (J1) were added to each vessel, containing now 0.5 g sediment (ww), 0.5 mL test solution and 0.5 mL bacterial suspension. The vessels were then incubated for 96 h. Six replicates per treatment group were set up for the test. In order to stop the assay, the nematodes were heat killed and stained with Rose Bengal. After extracting the nematodes from the sediment, body length and number of offspring were determined. A decrease in body length was found at 10 g/kg sediment (dw); no effect was found on reproduction up to the highest tested concentration of 10 g/kg sediment (dw). Therefore a NOEC of 5 g/kg sediment (dw) was derived from this study.

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 September 2018 to December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ISO standard 10872
Version / remarks:
2010 / Water quality -- Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda)
GLP compliance:
no
Remarks:
Testing house has longterm experience with the nematode test and participated in ring tests within the standardization process of ISO 10872
Analytical monitoring:
no
Details on sampling:
6 replicates/treatment group; 12 replicates in the control group;
10 test organisms/replicate
Vehicle:
no
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Artifical sediment according to ISO standard was used (41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content);
- Details of spiking: artifical sediment was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). The test substance was directly added to sediment.
- Equilibration time: 24 hours
- Equilibration conditions: 6 °C
- Controls: sediment, non-spiked
Test organisms (species):
nematodes
Details on test organisms:
Caenorhabditis elegans, strain N2; genotype: C. elegans wild type, DR subclone of CB original (Tc1 pattern);
Source: Caenorhabditis Genetic Centre; University of Minnesota, Minneapolis, USA;
first juvenile stage (J1) at start of the test (initial body length: Test 1: 315 (± 34) µm; Test 2: 332 (± 52) µm)
Cultivation of test organisms: according to ISO 10872
Study type:
laboratory study
Test type:
static
Type of sediment:
artificial sediment
Limit test:
no
Duration:
96 h
Test temperature:
19.9 – 20.4 °C
Nominal and measured concentrations:
0 (control), 0.1, 0.5, 1, 5, 10g/kg dry sediment (nominal), corresponding to 0, 0.06, 0.3, 0.6, 3 and 6 g/kg wet weight (nominal)
Details on test conditions:
TEST SYSTEM
- Test container: 12-well multidishes
- Sediment volume: 0 .300 (± 0.003) g of the dry spiked sediment or the controls were weighed into each test well
- Weight of wet sediment with and without water: 0.2 ml M9-medium (ISO 10872; 6 g Na2HPO4/L, 3 g KH2PO4/L, 5 g NaCl, 0.25 g MgSO4 7H2O/L) were added to 0.3 g dry sediment to moisten the sediment and to achieve a sediment wet weight of 0.5g and a water content of 40%

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 12
- Feeding: Immediately before the start of the test, 0.5 mL of bacterial suspension (Escherichia coli OP50 n M9-medium; cell density: 12,000 ± 600 FAU; according to ISO 10872) were added to the 0.5 g wet sediment and thoroughly mixed with a spatula, achieving 1 g of wet sediment with 0.300 (± 0.003) g dry weight.)

After 96 hours, all wells were stained with Bengal rose and heat treated at 80 °C for 10 minutes; afterwards stored at 8 °C (in accordance with ISO 10872)

EXTRACTION OF NEMATODES FROM TESTED SEDIMENTS
After termination of the test, test organisms and offspring are separated from the sediment by a flotation technique using Ludox TM50 (50 wt % suspension in water; Sigma-Aldrich; Batch: 07826PH; adjusted with water to a density of 1.13 g/mL). After mixing the sediment with Ludox-suspension and subsequent centrifugation, nematodes are found in the supernatant. After three extraction steps, the combined supernatant is analyzed. The added Ludox TM50 suspension has no impact on the test results, as it is added after the test has been terminated.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
10 other: g/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
5 other: g/kg dry sediment
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Details on results:
No significant effects on nematode growth or reproduction occurred up to and at 10 g/kg dry sediment.
Results with reference substance (positive control):
no reference substance used
Reported statistics and error estimates:
one-way ANOVA (post-hoc test: Dunnett’s test), descriptive statistics

Table:Validity criteria in controls; x = fulfilled

Validity Criteria

Test 1

Test 2

% Recovery in Controls (80 ≤ x ≤ 120%)

91.7 (x)

90.0 (x)

% Males in Controls

(≤ 10%)

0.0 (x)

0.0 (x)

% Fertility in Controls

(≥ 80%)

99.1 (x)

100.0 (x)

Reproduction in Controls (≥ 30 offspring / test organisms)

119 (x)

124 (x)

 

Validity criteria fulfilled:
yes
Conclusions:
A decrease in reproduction was found at 10 g/kg sediment (dw); no effect were found on growth.
Executive summary:

During the study artificial sediment (with particle size distribution of 41% sand (> 63 µm), 54.5% silt (2-63µm), 4.5% clay (<2µm) and 2.1% total organic content was spiked with the test substance in various concentrations (nominal concentration range between 0.5 and 10 g/kg sediment dry weight (dw)). For spiking of sediment the test substance was directly added to sediment. In order to allow the test substance to equally distribute between aqueous and solid phase, the spiked sediment was incubated for 24 h before the start of the test.

Test parameters were growth (body length in µm), and reproduction (number of offspring divided by number of introduced test organisms). Before the start of the assay, 0.5 mL of bacterial suspension (E. coli in M9 medium) were added to each vessel as food for the nematodes. After that 10 juvenile worms of the first stage (J1) were added to each vessel, containing now 0.5 g sediment (ww), 0.5 mL test solution and 0.5 mL bacterial suspension. The vessels were then incubated for 96 h. Six replicates per treatment group were set up for the test. In order to stop the assay, the nematodes were heat killed and stained with Rose Bengal. After extracting the nematodes from the sediment, body length and number of offspring were determined. No effects of treatment with the test substance were found up to and at 10 g/kg dry sediment (NOEC).

Endpoint:
sediment toxicity: short-term
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Description of key information

Due to the ubiquitous presence of silicon dioxide in the environment, harmful effects of synthetic amorphous silicas (SAS) on the benthic community are not to be expected. This is supported by the lack of long-term effects on growth and reproduction of nematodes with NOECs between 1 and 10 g/kg sediment (dw) for different types of SAS (pyrogenic, precipitated, gel, colloidal). Only at extremely high doses, exceeding guideline limit doses, were decreases in growth found (at 5-10 g/kg sediment (dw) with precipitated SAS, and at 10 g/kg sediment (dw) with pyrogenic SAS). A decrease in reproduction was found at 10 g/kg sediment (dw) for precipitated SAS and colloidal silica (40% suspension). No effects on growth or reproduction were found with silica gel at the highest tested dose of 10 g/kg sediment (dw). Therefore, an overall NOEC of 1 g/kg sediment (dw) can be derived from these studies.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:
1 000 mg/kg sediment dw

Additional information