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Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: BBA-Guideline: "Long-term toxicity test with Chironomus riparius: Development and validation of a new test system" pp70-79. Eds Streloke M and Kopp H.
Deviations:
no
Principles of method if other than guideline:
This study was carried out to determine the effects of the test substance on the development of Chironomus riparius in sediment-water system. Chironomus riparius were exposed to the test substance in laboratory sediment-water systems for 25 days at 20 ± 2°C at concentrations ranging from 31.25 to 2000 µg/L. After 10 days, the test systems were observed daily for emerging adults and numbers of males and females were recorded. The test was terminated 25 days after application.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Overlying Water Phase:
i) On days 0, 7 and 25, overlying water from the test concentrations was sampled (110 ml). A subsample was mixed 50:50 with acetonitrile, passed through a 0.45 µm Millex® filter into glass vials and analyzed directly by HPLC.
ii) If test substance levels were below the level of detection using the above method, then a further subsample of the overlying water was concentrated prior to analysis by solid-phase extraction using a C18 Empore® disc by the following method:
The disc was first conditioned as follows:
10 ml acetonitrile drawn through the disc under vacuum and the disc dried.
10 ml methanol, drawn through the disc but leaving a meniscus above the disc.
10 ml ultra-pure water, drawn through the disc and leaving a meniscus above the disc.

An overlying water sample was centrifuged to remove particulate matter. The supernatant (100 ml) was modified with 1 ml methanol, drawn through the disc under vacuum and then dried for approximately 6 minutes. The test substance was eluted with 4 x 5 ml acetonitrile, which was evaporated to dryness by rotary evaporation under vacuum, and re-dissolved in 50:50 acetonitrile:water for analysis by HPLC.

Sediment Phase:
On day 25, after removal of the remaining overlying water, the sediment was mixed by stirring and a subsample taken (approximately 40 g wet weight), and transferred to Nalgene® centrifuge bottles (250 ml). The sediment was sequentially extracted with 2 x 75 ml acetonitrile by shaking on a reciprocal shaker for 30 minutes, centrifuging for 5 minutes at 1625 G, and then removing the supernatant. The extracts were combined and made up to 200 ml with acetonitrile. An aliquot (1 ml) was mixed 50:50 with ultra-pure water and analyzed by HPLC to determine the concentration of the test substance. If levels test substance were below the limit of detection by analyzing 1 mL of extract, a further 20 ml of extract was subsampled, evaporated to dryness by rotary evaporation, re-dissolved in 50:50 acetonitrile:water and re-analyzed as before.
Vehicle:
yes
Remarks:
methanol
Details on sediment and application:
The artificial sediment used was prepared in accordance with OECD guideline No. 207. It was a mixture of the following ingredients (on the basis of dry weights): 70% fine silica sand; 20% kaolinite clay; 10% peat (air dried and finely ground prior to mixing); CaCO3 at 5 g/kg. The organic carbon content of the sediment was measured at 4.2%.

25 first instar larvae (2 day old) were introduced into each test vessel. These were selected at random and introduced to the test vessels, below the water surface, using a narrow-mouthed (2 mm diameter) pipette. Prior to this and for 24 hours after the addition of the larvae, aeration was stopped.

24 hours after adding the larvae, the test chemical was applied to the water column. Based on preliminary studies, a series of application solutions in methanol were prepared to give the nominal concentrations in the overlying water, spiking in a 250 µl aliquot.

A control and a solvent control (spiked with 250 µl methanol) were prepared as for the treated systems. The chemical was introduced just below the water surface, using a pipette, and gently mixed to ensure homogenous distribution but without disturbance of the sediment.
Test organisms (species):
Chironomus riparius
Details on test organisms:
Chironomus riparius is a member of the widespread dipteran insect family Chironomidae, commonly referred to as non-biting midges. It has four sediment-dwelling larval instars which are widely used in sediment toxicity testing. The organisms used in this study were obtained from laboratory cultures at Jealott's Hill Research Station. Mixed age cultures were maintained in hard water at approximately 20°C on a 16 hour 8 hour cycle. Culture vessels were 5 litre plastic aquaria containing water approximately 12 cm deep overlying approximately 2 cm of silver sand. Cultures were fed ground Tetramin® (high protein fish food) as required.

For the test, egg ropes were removed from the cultures and hatched in small plastic trays. On the day of hatching, the larvae were transferred to large glass crystallizing dishes containing hard water overlying a thin layer of silver sand. Gentle aeration was provided and Chlorella vulgaris and ground Tetramin® were added as food. After 2 days, the first instar C. riparius were removed for use in the test by gentle agitation of the crystallizing dish and decanting off the water containing the larvae into small plastic trays for counting out into the test vessels.
Study type:
laboratory study
Type of sediment:
artificial sediment
Duration:
25 d
Exposure phase:
larvae from first generation (P)
Hardness:
178 mg CaCO3/L (used in the study)
129-154 mg CaCO3/L (at the end of the study)
Test temperature:
water-bath throughout the study was 20.9°C, ranging from 18.1 to 21.6°C and in a test vessel 21.0°C, ranging from 19.2 to 21.6°C
pH:
7.6-8.5
Dissolved oxygen:
≥6.7 mg/L
Conductivity:
490-510 µS/cm
Nominal and measured concentrations:
Nominal: 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/L
Measured (on day 25): 0.008, 0.023, 0.054, 0.24, 1.7, 4.1, 8.8 mg/kg
Details on test conditions:
TEST SYSTEM
- Test container: 3000 L glass beakers, 13 cm diameter, containing a 2 cm layer of sediment and approximately 18 cm depth (2.5 L) of overlying water
- Sediment volume: 300 g when moist, equivalent to 267 g dry weight
- Aeration: yes

EXPOSURE REGIME
- No. of organisms per container (treatment): 25
- No. of replicates per treatment group: 3
- No. of replicates per control / vehicle control: 3

CHARACTERIZATION OF (ARTIFICIAL) SEDIMENT
- pH: 5.8
- % sand (2000-50 µm): 77
- % silt 50-2 µm): 9
- % clay (<2 µm): 14
- Organic carbon (%): 4.2
- Organic matter (%): 7.3
- CEC: 12.9 meq/100 g
- Classification: Sandy loam

OTHER TEST CONDITIONS
- Photoperiod: 16 hour light : 8 hour dark cycle
- Light intensity: Approximately 700 lux

VEHICLE CONTROL PERFORMED: Yes
Reference substance (positive control):
no
Key result
Duration:
25 d
Dose descriptor:
EC50
Effect conc.:
140 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
emergence rate
Remarks on result:
other: 95% CI: 118-165 µg/L
Key result
Duration:
25 d
Dose descriptor:
NOEC
Effect conc.:
62.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
emergence rate
Details on results:
Emergence began on day 11 in untreated controls, solvent controls and test concentrations up to and including 125 µ/L and on day 12 in 250 µ/L. On day 14 in one test chamber at 500 µg/L one adult emerged but no further emergence occurred at this test concentration. There was no emergence in the top two test concentrations. Total emergence in the untreated and solvent controls was 96 and 100% of the larvae introduced, respectively. It was similar in the test concentrations up to and including 62.5 µg/L, ranging from 93 to 96%. At 125, 250 and 500 µg/L, emergence was 64, 15 and 1% respectively. Examination of the sediments from the replicates where 100% emergence had not occurred on day 25 revealed no live larvae.

The highest dose levels, 1000 and 2000 µg/L, showed zero emergence and were therefore not included in the statistical analyses. The EC50 on total emergence (the concentration reducing total emergence by 50%) based on the concentrations applied to the water at day 0 was 140 µg/L , with a 95% confidence interval of 118 to 165 µg/L. The NOEC based on total emergence was 62.5 µg/L. The statistical analysis to determine the NOEC based on time to emergence did not include the 500 µg/L dose level as only 1 midge emerged in the combined replicates. None of the remaining treatments showed a significant adverse effect on time to emergence compared to the combined controls.
Validity criteria fulfilled:
yes
Conclusions:
EC50: 140 µg/L (total emergence)
NOEC: 62.5 µg/L (total emergence)
Executive summary:

Chironomus riparius were exposed to the test substance in laboratory sediment-water systems (267 g dry weight sediment and 2.5 L overlying water) for 25 days at 20 ± 2°C. An artificial sediment was used with an organic carbon content of 4.2%. Test systems were treated at concentrations ranging from 31.25 to 2000 µg/L by applying the chemical to the water phase of the system, which already contained first instar C. riparius. After 10 days, the test systems were observed daily for emerging adults and numbers of males and females were recorded. The test was terminated 25 days after application.

Sampled one hour after application, measured test substance concentrations in the water phase ranged from 77 to 101% of nominal. These had declined to between 3 and 63% of nominal by day 7 and between 1 and 46 % by day 25. Measured concentrations of extractable test substance residues in sediment on day 25 ranged from 0.008 mg/kg at the lowest concentration, to 8.8 mg/kg at the highest.

The EC50 on total emergence (the concentration reducing total emergence by 50%) based on the concentrations applied to the water at day 0 was 140 µg/L. The NOEC based on total emergence was 62.5 µg/L. There was no significant adverse effect on time to emergence.

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chironomus riparius larvae were exposed to the test substance in laboratory sediment-water systems at 20 ± 2°C at concentrations ranging from 1.25 to 80 mg/kg. After 10 days, the test systems were observed daily for emerging adults and numbers of males and females were recorded. The test was terminated 28 days after application.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Overlying Water Phase:
i) At day 0, overlying water of replicate D was sampled (110 ml). The water samples were allowed to settle, and a subsample of the supernatant was mixed 50:50 with acetonitrile and analyzed directly by HPLC.
ii) At day 28, the overlying water of replicate C was sampled (110 ml) and centrifuged to settle particulate matter. The supernatant was subsampled, mixed 50:50 with acetonitrile and analyzed directly by HPLC. However, the level of test substance in the water of the lowest test concentration (1.25 mg/kg) was below the limit of detection by this method. Therefore, a further subsample of this concentration, the control and solvent control were concentrated prior to analysis by solid-phase extraction using a C18 Empore® disc by the following method:
The disc was first conditioned as follows:
10 ml acetonitrile drawn through the disc under vacuum and the disc dried.
10 ml methanol, drawn through the disc but leaving a meniscus above the disc.
10 ml ultra-pure water, drawn through the disc and leaving a meniscus above the disc.

The supernatant of the overlying water samples (100 ml) was modified with 1 ml methanol, drawn through the disc under vacuum and then dried for approximately 6 minutes. The test substance was eluted with 4 x 5 ml acetonitrile, which was evaporated to dryness by rotary evaporation under vacuum, and re-dissolved in 50:50 acetonitrile:water for analysis by HPLC.

Sediment Phase:
After removal of the remaining overlying water, the sediment was transferred to Nalgene® centrifuge bottles (250 ml). Acetonitrile (75 ml) was used to rinse the residual sediment from each test jar into the centrifuge bottles. The sediment was extracted by shaking on a reciprocal shaker for 30 minutes, centrifuging for 5 minutes at 1625 G. The supernatant was removed to volumetric flasks and the extraction procedure was repeated with further acetonitrile (75 ml). The extracts were combined and made up to 200 ml with acetonitrile. An aliquot (1 ml) was mixed 50:50 with ultra-pure water and analyzed by HPLC to determine the concentration of the test substance.
Vehicle:
yes
Remarks:
methanol
Details on sediment and application:
The artificial sediment used was prepared in accordance with OECD guideline No. 207.
It was a mixture of the following ingredients (on the basis of dry weights): 70% fine silica sand; 20% kaolinite clay; 10% peat (air dried and finely ground prior to mixing); CaCO3 at 5 g/kg.
The organic carbon content of the sediment was measured at 4.2%.
Test organisms (species):
Chironomus riparius
Details on test organisms:
Chironomus riparius is a member of the widespread dipteran insect family Chironomidae, commonly referred to as non-biting midges. It has four sediment-dwelling larval instars which are widely used in sediment toxicity testing. The organisms used in this study were obtained from laboratory cultures at Jealott's Hill Research Station. Mixed age cultures were maintained in hard water at approximately 20°C on a 16 hour 8 hour cycle. Culture vessels were 5 litre plastic aquaria containing water approximately 12 cm deep overlying approximately 2 cm of silver sand. Cultures were fed ground Tetramin® (high protein fish food) as required.

To obtain organisms for the test, egg ropes were removed from the cultures and hatched in small plastic trays. On the day of hatching, the larvae were transferred to large glass crystallizing dishes containing hard water overlying a thin layer of silver sand. Gentle aeration was provided and Chlorella vulgaris and ground Tetramin® were added as food. After 2 days, 20 x the first instar C. riparius were added to each test vessel of replicates A, B, and C.
Study type:
laboratory study
Type of sediment:
artificial sediment
Duration:
28 d
Exposure phase:
larvae from first generation (P)
Hardness:
178 mg CaCO3/L (used in the study)
75-100 mg CaCO3/L (at the end of the study)
Test temperature:
water-bath throughout the study was 19.7°C, ranging from 19.2 to 20.6°C and in a test vessel 20.0°C, ranging from 17.8 to 20.9°C
pH:
Day 0: 8.0-8.3
Day 28: 5.7-8.1
Dissolved oxygen:
8.3-8.9 mg/L
Conductivity:
Day 0: 380-390 µS/cm
Day 28: 390-500 µS/cm
Nominal and measured concentrations:
Nominal: 1.25, 2.5, 5, 10, 20, 40, 80 mg/kg
Measured (on day 28): 0.004, 0.010, 0.026, 0.057, 0.17, 0.41, 0.83 mg/L (overlying water); 0.96, 2.0, 4.2, 7.6, 17, 36, 70 mg/kg (sediment)
Details on test conditions:
TEST SYSTEM
- Test container: 500 mL glass jars
- Sediment volume: 30 g dry weight sediment and 250 ml water
- Aeration: yes

EXPOSURE REGIME
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: 3
- No. of replicates per control / vehicle control: 3

CHARACTERIZATION OF (ARTIFICIAL) SEDIMENT
- pH: 5.8
- % sand (2000-50 µm): 77
- % silt 50-2 µm): 9
- % clay (<2 µm): 14
- Organic carbon (%): 4.2
- Organic matter (%): 7.3
- CEC: 12.9 meq/100 g
- Classification: Sandy loam

OTHER TEST CONDITIONS
- Photoperiod: 16 hour light : 8 hour dark cycle
- Light intensity: Approximately 700 lux

VEHICLE CONTROL PERFORMED: Yes
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
19 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
emergence rate
Remarks on result:
other: 95% CI: 12-59 mg/kg
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
5 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
emergence rate
Details on results:
Emergence began on day 13 in the control and measured test concentration 2.2 mg/kg, day 14 in measured test concentrations up to 9.1 mg/kg and day 15 in 20 mg/kg. No adults emerged in the top two concentrations, 40 and 77 mg/kg. The last adult to emerge during the study was on day 26 from an untreated control. The days of last emergence in the solvent control, 1.2, 2.2, 5.0, 9.1 and 20 mg/kg were 25, 20, 23, 20, 24 and 20 respectively. No live larvae were found on examination of the sediments on day 28. The highest dose levels, 40 and 77 mg/kg, showed zero emergence and were therefore not included in the statistical analyses. The EC50 for total emergence (the concentration reducing total emergence by 50%) based upon measured sediment concentrations at day 0 was 19 mg/kg, with 95% confidence intervals of 12 to 59 mg/kg. The NOEC based on total emergence was 5.0 mg/kg. There was no significant adverse effect on time to emergence between the combined controls and the test concentrations analyzed.
Validity criteria fulfilled:
yes
Conclusions:
EC50: 19 mg/kg (total emergence)
NOEC: 5 mg/kg (total emergence)
Executive summary:

Chironomus riparius larvae were exposed to test substance in laboratory sediment-water systems at 20 ± 2°C using an artificial sediment with a measured organic carbon content of 4.2%. Test systems were prepared by applying the chemical to a sediment-water slurry (30 g dry weight sediment : 250 ml water), shaking and then rolling for two hours. After allowing the system to settle for two days, first instar C. riparius were introduced into the sediment-water systems (day 0). After 10 days, the test systems were observed daily for emerging adults, and the numbers of males and females were recorded. The test was terminated after 28 days. The distribution of the chemical between the sediment and overlying water was determined at the time of introduction of the organisms and at the end of the study.

Test substance concentrations measured in the sediment on day 0 ranged from 1.2 to 77 mg/kg, and from 0.96 to 70 mg/kg when measured on day 28. Measured concentrations of test substance in the overlying water on day 0 ranged from 0.006 to 1.3 mg/L and at day 28 from 0.004 to 0.83 mg/L.

The EC50 based on total emergence and measured sediment concentrations at day 0 was 19 mg/kg. The NOEC based on total emergence was 5 mg/kg. There was no significant adverse effect on time to emergence in the test concentrations analyzed, compared with the combined controls.

Description of key information

21-day NOEC (Chironomus riparius): 0.0084 mg a.s./L (water spiked; based on emergence); BBA-Guideline: "Long-term toxicity test with Chironomus riparius: Development and validation of a new test system

; Reliability = 2

28-day NOEC (Chironomus riparius): 5 mg a.s./kg sediment (sediment spiked; based on emergence); no guideline; Reliability = 1

Key value for chemical safety assessment

Additional information