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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February to March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were taken to confirm test material presence in test solutions and absence in control. Samples were taken from each test and control solution on Days 0, 7, 14, and 20 and the corresponding old solutions (three individual replicates) on Days 1, 8, 15 and 21, unless complete mortality occurred prior to the sampling event. Twenty-four samples from each group were analyzed during the twenty-one day study.
Vehicle:
no
Details on test solutions:
The exposure solutions were generated by aerating the test substance alone in a closed system using air stones at an air flow rate of 45 +/-5 cc/min. The vapor generated of the test substance from the aeration procedure flowed into aspirator bottles and bubbled through vehicle/dilution medium (reconstituted water). The test substance saturated vapor passed through air stones near the bottom of the aspirator bottles providing maximum contact between the test substance in the vapor phase and the vehicle/dilution medium. The systems were allowed to aerate/equilibrate for one day prior to preparing the exposure solutions for the first time. Ten replicates per treatment were tested.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test organisms were cultured at the test facility. The original culture was supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado, USA and received at the test facility on 11 April 2002.
For the test, eight adult daphnids were kept in 1-liter glass culture beakers with approximately 800 mL of reconstituted water (study vehicle/dilution water). The culture chamber was maintained at 20 ± 1 degrees C under a 16 hour light 8 hour dark photoperiod (10 - 20 foot/candles, 108 - 215 Lux). Day 0 cultures were started daily (at least five days per week) using eight <24 hour old neonates from culture beakers between 12 and 18 days old, exhibiting ≤20% adult mortality. Cultures were transferred to fresh reconstituted water on regular intervals to ensure that ≤24 hour old neonates are available for studies and to start new cultures.
Cultures of Daphnia magna were fed Pseudokirchneriella subcapitata (approximately 4.5 x 105 cells/mL) and 4.0 mL of a yeast / salmon starter / wheat grass (YTC) mixture per 800 mL daily (five days per week at a minimum). YTC and algae were supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado, USA.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
The hardness of the dilution water during the study was 150 mg/L as CaCO3.
Test temperature:
19.2 to 19.9 degrees C for test substance systems.
19.2 to 19.9 degrees C for control systems.
pH:
8.0 to 8.6 for test substance systems.
8.0 to 8.6 for control systems.
Dissolved oxygen:
7.9 to 9.1 mg/L for test substance systems.
7.9 to 9.1 mg/L for control systems.
Nominal and measured concentrations:
Nominal loading rates were control, 0.026, and 0.085 mg/L, which measured 0.0, 0.025, and 0.050 mg/L.
Details on test conditions:
Ten replicates per treatment were tested. One <24 hour old daphnid was added to each replicate. The adults were transferred to fresh solution daily. The test chambers were 125 mL capacity clear glass bottles containing approximately 125 mL of solution (no headspace). The test chambers were closed with foil lined screw type lids to minimize contamination, evaporation, and/or volatilization. A separate set of test chambers was used on a rotating basis for renewals. Feed was first added to each test chamber. Test chambers were filled with the appropriate amount of vehicle/dilution water. The adult Daphnia magna from the old test solution were transferred to the vehicle/dilution water using a glass pipette.
Diurnal light: approximately 16 hours light and 8 hours dark. Daylight intensity ranged from approximately 16 to 19 µE•m-2•s-1 during full daylight periods of the study.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.025 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
The test substance also showed no effects based on growth at 0.025 mg/L.
Details on results:
The following are the endpoint results for the control and test solutions:
Adult Neonates Adult
Group Group Immobilization per Ave. Length
(nominal) (meas.) (%) Adult (mm)
Control 0.0 µg/L 0 119 4.6
26 µg/L 25 µg/L 0 119 4.4
85 µg/L 50 µg/L 100 n/a n/a

The control daphnids released their first brood between days 7 and 10. The coefficient of variation (cv) for control fecundity was 16%, the guideline indicates that the cv for control reproduction in a “well run” study should be ≤25%. The test was considered acceptable as less than 20% of control organisms were immobilized and mean reproduction in the controls exceeded 60 young per adult.
Results with reference substance (positive control):
None used
Reported statistics and error estimates:
The chronic survival and reproduction NOEC for each test substance was based on empirical observation. The length NOEC was determined using the Bonferroni t-test using TOXSTAT software.
Validity criteria fulfilled:
yes
Conclusions:
The test substance showed no effects on survival, reproduction, or length at nominal and measured concentrations of 0.026 and 0.025 mg/L, respectively. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) and No Observed Effect Concentration (NOEC) values are 0.026 and 0.025 mg/L, respectively, for these endpoints based upon nominal loading rates and measured (geometric means) concentrations.
Executive summary:

The test substance showed no effects on survival, reproduction, or length at nominal and measured concentrations of 0.026 and 0.025 mg/L, respectively. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) and No Observed Effect Concentration (NOEC) values are 0.026 and 0.025 mg/L, respectively, for these endpoints based upon nominal loading rates and measured (geometric means) concentrations. The next highest loading rate and concentration tested, 0.085 and 0.050 mg/L, respectively, produced 100% immobilization of the adults exposed.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
February to March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were taken to confirm test material presence in test solutions and absence in control. Samples were taken from each test and control solution on Days 0, 7, 14, and 20 and the corresponding old solutions (three individual replicates) on Days 1, 8, 15 and 21, unless complete mortality occurred prior to the sampling event. Twenty-four samples from each group were analyzed during the twenty-one day study.
Vehicle:
no
Details on test solutions:
The exposure solutions were generated by aerating the test substance alone in a closed system using air stones at an air flow rate of 45 +/-5 cc/min. The vapor generated of the test substance from the aeration procedure flowed into aspirator bottles and bubbled through vehicle/dilution medium (reconstituted water). The test substance saturated vapor passed through air stones near the bottom of the aspirator bottles providing maximum contact between the test substance in the vapor phase and the vehicle/dilution medium. The systems were allowed to aerate/equilibrate for one day prior to preparing the exposure solutions for the first time. Ten replicates per treatment were tested.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test organisms were cultured at the test facility. The original culture was supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado, USA and received at the test facility on 11 April 2002.
For the test, eight adult daphnids were kept in 1-liter glass culture beakers with approximately 800 mL of reconstituted water (study vehicle/dilution water). The culture chamber was maintained at 20 ± 1 degrees C under a 16 hour light 8 hour dark photoperiod (10 - 20 foot/candles, 108 - 215 Lux). Day 0 cultures were started daily (at least five days per week) using eight <24 hour old neonates from culture beakers between 12 and 18 days old, exhibiting ≤20% adult mortality. Cultures were transferred to fresh reconstituted water on regular intervals to ensure that ≤24 hour old neonates are available for studies and to start new cultures.
Cultures of Daphnia magna were fed Pseudokirchneriella subcapitata (approximately 4.5 x 105 cells/mL) and 4.0 mL of a yeast / salmon starter / wheat grass (YTC) mixture per 800 mL daily (five days per week at a minimum). YTC and algae were supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado, USA.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
The hardness of the dilution water during the study was 150 mg/L as CaCO3.
Test temperature:
19.2 to 19.9 degrees C for test substance systems.
19.2 to 19.9 degrees C for control systems.
pH:
8.0 to 8.6 for test substance systems.
8.0 to 8.6 for control systems.
Dissolved oxygen:
7.9 to 9.1 mg/L for test substance systems.
7.9 to 9.1 mg/L for control systems.
Nominal and measured concentrations:
Nominal loading rates were control, 0.026, and 0.085 mg/L, which measured 0.0, 0.025, and 0.050 mg/L.
Details on test conditions:
Ten replicates per treatment were tested. One <24 hour old daphnid was added to each replicate. The adults were transferred to fresh solution daily. The test chambers were 125 mL capacity clear glass bottles containing approximately 125 mL of solution (no headspace). The test chambers were closed with foil lined screw type lids to minimize contamination, evaporation, and/or volatilization. A separate set of test chambers was used on a rotating basis for renewals. Feed was first added to each test chamber. Test chambers were filled with the appropriate amount of vehicle/dilution water. The adult Daphnia magna from the old test solution were transferred to the vehicle/dilution water using a glass pipette.
Diurnal light: approximately 16 hours light and 8 hours dark. Daylight intensity ranged from approximately 16 to 19 µE•m-2•s-1 during full daylight periods of the study.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.025 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
The test substance also showed no effects based on growth at 0.025 mg/L.
Details on results:
The following are the endpoint results for the control and test solutions:
Adult Neonates Adult
Group Group Immobilization per Ave. Length
(nominal) (meas.) (%) Adult (mm)
Control 0.0 µg/L 0 119 4.6
26 µg/L 25 µg/L 0 119 4.4
85 µg/L 50 µg/L 100 n/a n/a

The control daphnids released their first brood between days 7 and 10. The coefficient of variation (cv) for control fecundity was 16%, the guideline indicates that the cv for control reproduction in a “well run” study should be ≤25%. The test was considered acceptable as less than 20% of control organisms were immobilized and mean reproduction in the controls exceeded 60 young per adult.
Results with reference substance (positive control):
None used
Reported statistics and error estimates:
The chronic survival and reproduction NOEC for each test substance was based on empirical observation. The length NOEC was determined using the Bonferroni t-test using TOXSTAT software.
Validity criteria fulfilled:
yes
Conclusions:
The test substance showed no effects on survival, reproduction, or length at nominal and measured concentrations of 0.026 and 0.025 mg/L, respectively. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) and No Observed Effect Concentration (NOEC) values are 0.026 and 0.025 mg/L, respectively, for these endpoints based upon nominal loading rates and measured (geometric means) concentrations.
Executive summary:

The test substance showed no effects on survival, reproduction, or length at nominal and measured concentrations of 0.026 and 0.025 mg/L, respectively. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) and No Observed Effect Concentration (NOEC) values are 0.026 and 0.025 mg/L, respectively, for these endpoints based upon nominal loading rates and measured (geometric means) concentrations. The next highest loading rate and concentration tested, 0.085 and 0.050 mg/L, respectively, produced 100% immobilization of the adults exposed.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - September 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for analysis of new and old test solutions and controls were taken on days 1, 2, 3, 7, 10, 14, 17, and 21.
Vehicle:
no
Details on test solutions:
The water accommodated fraction (WAF) of the test material was prepared by stirring the test material in the exposure solution for approximately 2 days with a 1 to 3 hour settling period, after which the aqueous phase was removed and added to the test system. An equilibration study was conducted over a period of 96 hours prior to the definitive study to determine the approximate length of time needed to stir the test substance in the test media to acheive a saturated solution. Results suggested that a stirring period of approximately 2 days would be sufficient to reach equilibrium. The stirring period to develop WAFs used through the study ranged from 44.5 to 46.5 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
Organisms used in the test were from a laboratory culture were less than 24 hour old. The source of the culture was from Zeneca Brixham Laboratory from I.R.Ch.A., France.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
154 to 170 mg/L CaCO3
Test temperature:
20.1 to 20.5 degrees C
pH:
8.3 to 9.0
Dissolved oxygen:
7.8 to 9.1 mg/L
Nominal and measured concentrations:
The nominal treatment levels of the test substance included a control and 1000 mg/L, which measured <0.002 (the limit of detection, with the exception of the day 10 control sample, which measured 0.003 mg/L) and 0.036 mg/L, respectively. The measured data represent the mean test substance concentration of samples taken from new and 24-hour old treatment solutions from days 1, 2, 3, 7, 10, 14, 17, and 21. The mean concentration of the test substance in new and old WAFs was 0.047 and 0.025 mg/L, respectively. The percent decrease in concentration of test material in solution from new to old WAFs on the sampling days ranged from 41 to 54%.
Details on test conditions:
The study was conducted in closed test systems that were renewed daily during the study. The test systems used were 150 ml glass Erlenmeyer flasks that were completely filled with media so as not to leave any headspace. The control and treatment level were each evaluated in 10 replicate test systems. One organism, less than 24 hours old, was added to each control and treatment solution flasks and were fed each day of the study. The feed was chlorella vulgaris at a rate of 0.3 mg carbon/daphnid/day. This rate was greater than that suggested in the OECD guideline (0.2 mg carbon/daphnid/day) because the test vessels were larger than specified by the guideline (50 to 100 ml). Light intensity was 505 lux.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Adult immobility: control - 10%; 1 mg/L - 10%
Total live young produced: control - 1245; 1 mg/L - 841
Mean number of live young produced per surviving adult: control - 138; 1 mg/L - 93
Mean number of dead young produced per surviving adult: control - 0; 1 mg/L - 13

Reduction in fecundity in 1 mg/L treatment relative to control:
Brood 1 - 88%
Brood 2 - 73%
Brood 3 - 39%
Brood 4 - ca.0%
Brood 5 - (-46%) the treatment solution produced a greater number of live young (31) than the control (16 live young)
Reported statistics and error estimates:
The significance of differences in the mean number of live young produced by adults in 21 days between the control and treatment solutions was assessed using a two-sample T-test.
Validity criteria fulfilled:
yes
Remarks:
The results show that survival of the adult control daphnids (90%) and the mean number of living offspring produced per surviving control parent (138) fulfilled the test validity criteria of >/= 80% and >/= 60 young/daphnia.
Conclusions:
During the 21-day study, the adult daphnids exposed to the test substance at a loading rate of 1 mg/L produced significantly fewer (<0.05) live offspring per adult than the adult daphnids in the control media. Therefore, the 21-day NOELR for reproduction was <1 mg/L. There was 10% immobility of parents in the treatment solution, which was equivalent to the control mortality, therefore the EL50 can be reported as >1 mg/L.
Executive summary:

During the 21-day study, the adult daphnids exposed to the test substance at a loading rate of 1 mg/L produced significantly fewer (<0.05) live offspring per adult than the adult daphnids in the control media. Therefore, the 21-day NOELR for reproduction was <1 mg/L. There was also a greater number of dead offspring produced by the daphnids exposed to the test substance compared with those in the control. There was 10% immobility of parents in the treatment solution, which was equivalent to the control mortality, therefore the EL50 can be reported as >1 mg/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
August - September 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for analysis of new and old test solutions and controls were taken on days 1, 2, 3, 7, 10, 14, 17, and 21.
Vehicle:
no
Details on test solutions:
The water accommodated fraction (WAF) of the test material was prepared by stirring the test material in the exposure solution for approximately 2 days with a 1 to 3 hour settling period, after which the aqueous phase was removed and added to the test system. An equilibration study was conducted over a period of 96 hours prior to the definitive study to determine the approximate length of time needed to stir the test substance in the test media to acheive a saturated solution. Results suggested that a stirring period of approximately 2 days would be sufficient to reach equilibrium. The stirring period to develop WAFs used through the study ranged from 44.5 to 46.5 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
Organisms used in the test were from a laboratory culture were less than 24 hour old. The source of the culture was from Zeneca Brixham Laboratory from I.R.Ch.A., France.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
154 to 170 mg/L CaCO3
Test temperature:
20.1 to 20.5 degrees C
pH:
8.3 to 9.0
Dissolved oxygen:
7.8 to 9.1 mg/L
Nominal and measured concentrations:
The nominal treatment levels of the test substance included a control and 1000 mg/L, which measured <0.002 (the limit of detection, with the exception of the day 10 control sample, which measured 0.003 mg/L) and 0.036 mg/L, respectively. The measured data represent the mean test substance concentration of samples taken from new and 24-hour old treatment solutions from days 1, 2, 3, 7, 10, 14, 17, and 21. The mean concentration of the test substance in new and old WAFs was 0.047 and 0.025 mg/L, respectively. The percent decrease in concentration of test material in solution from new to old WAFs on the sampling days ranged from 41 to 54%.
Details on test conditions:
The study was conducted in closed test systems that were renewed daily during the study. The test systems used were 150 ml glass Erlenmeyer flasks that were completely filled with media so as not to leave any headspace. The control and treatment level were each evaluated in 10 replicate test systems. One organism, less than 24 hours old, was added to each control and treatment solution flasks and were fed each day of the study. The feed was chlorella vulgaris at a rate of 0.3 mg carbon/daphnid/day. This rate was greater than that suggested in the OECD guideline (0.2 mg carbon/daphnid/day) because the test vessels were larger than specified by the guideline (50 to 100 ml). Light intensity was 505 lux.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Adult immobility: control - 10%; 1 mg/L - 10%
Total live young produced: control - 1245; 1 mg/L - 841
Mean number of live young produced per surviving adult: control - 138; 1 mg/L - 93
Mean number of dead young produced per surviving adult: control - 0; 1 mg/L - 13

Reduction in fecundity in 1 mg/L treatment relative to control:
Brood 1 - 88%
Brood 2 - 73%
Brood 3 - 39%
Brood 4 - ca.0%
Brood 5 - (-46%) the treatment solution produced a greater number of live young (31) than the control (16 live young)
Reported statistics and error estimates:
The significance of differences in the mean number of live young produced by adults in 21 days between the control and treatment solutions was assessed using a two-sample T-test.
Validity criteria fulfilled:
yes
Remarks:
The results show that survival of the adult control daphnids (90%) and the mean number of living offspring produced per surviving control parent (138) fulfilled the test validity criteria of >/= 80% and >/= 60 young/daphnia.
Conclusions:
During the 21-day study, the adult daphnids exposed to the test substance at a loading rate of 1 mg/L produced significantly fewer (<0.05) live offspring per adult than the adult daphnids in the control media. Therefore, the 21-day NOELR for reproduction was <1 mg/L. There was 10% immobility of parents in the treatment solution, which was equivalent to the control mortality, therefore the EL50 can be reported as >1 mg/L.
Executive summary:

During the 21-day study, the adult daphnids exposed to the test substance at a loading rate of 1 mg/L produced significantly fewer (<0.05) live offspring per adult than the adult daphnids in the control media. Therefore, the 21-day NOELR for reproduction was <1 mg/L. There was also a greater number of dead offspring produced by the daphnids exposed to the test substance compared with those in the control. There was 10% immobility of parents in the treatment solution, which was equivalent to the control mortality, therefore the EL50 can be reported as >1 mg/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February to March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were taken to confirm test material presence in test solutions and absence in control. Samples were taken from each test and control solution on Days 0, 7, 14, and 20 and the corresponding old solutions (three individual replicates) on Days 1, 8, 15 and 21, unless complete mortality occurred prior to the sampling event. Twenty-four samples from each group were analyzed during the twenty-one day study.
Vehicle:
no
Details on test solutions:
The exposure solutions were generated by aerating the test substance alone in a closed system using air stones at an air flow rate of 45 +/-5 cc/min. The vapor generated of the test substance from the aeration procedure flowed into aspirator bottles and bubbled through vehicle/dilution medium (reconstituted water). The test substance saturated vapor passed through air stones near the bottom of the aspirator bottles providing maximum contact between the test substance in the vapor phase and the vehicle/dilution medium. The systems were allowed to aerate/equilibrate for one day prior to preparing the exposure solutions for the first time. Ten replicates per treatment were tested.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test organisms were cultured at the test facility. The original culture was supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado, USA and received at the test facility on 11 April 2002.
For the test, eight adult daphnids were kept in 1-liter glass culture beakers with approximately 800 mL of reconstituted water (study vehicle/dilution water). The culture chamber was maintained at 20 ± 1 degrees C under a 16 hour light 8 hour dark photoperiod (10 - 20 foot/candles, 108 - 215 Lux). Day 0 cultures were started daily (at least five days per week) using eight <24 hour old neonates from culture beakers between 12 and 18 days old, exhibiting ≤20% adult mortality. Cultures were transferred to fresh reconstituted water on regular intervals to ensure that ≤24 hour old neonates are available for studies and to start new cultures.
Cultures of Daphnia magna were fed Pseudokirchneriella subcapitata (approximately 4.5 x 105 cells/mL) and 4.0 mL of a yeast / salmon starter / wheat grass (YTC) mixture per 800 mL daily (five days per week at a minimum). YTC and algae were supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado, USA.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
The hardness of the dilution water during the study was 150 mg/L as CaCO3.
Test temperature:
19.2 to 19.9 degrees C for test substance systems.
19.2 to 19.9 degrees C for control systems.
pH:
8.0 to 8.6 for test substance systems.
8.0 to 8.6 for control systems.
Dissolved oxygen:
7.8 to 9.2 mg/L for test substance systems.
7.9 to 9.1 mg/L for control systems.
Nominal and measured concentrations:
Nominal loading rates were control, 0.020, and 0.040 mg/L, which measured 0.0, 0.011, and 0.015 mg/L.
Details on test conditions:
Ten replicates per treatment were tested. One <24 hour old daphnid was added to each replicate. The adults were transferred to fresh solution daily. The test chambers were 125 mL capacity clear glass bottles containing approximately 125 mL of solution (no headspace). The test chambers were closed with foil lined screw type lids to minimize contamination, evaporation, and/or volatilization. A separate set of test chambers was used on a rotating basis for renewals. Feed was first added to each test chamber. Test chambers were filled with the appropriate amount of vehicle/dilution water. The adult Daphnia magna from the old test solution were transferred to the vehicle/dilution water using a glass pipette.
Diurnal light: approximately 16 hours light and 8 hours dark. Daylight intensity ranged from approximately 16 to 19 µE•m-2•s-1 during full daylight periods of the study.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.011 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
The test substance also showed no effects based on growth at 0.011 mg/L.
Details on results:
The following are the endpoint results for the control and test solutions:
Adult Neonates Adult
Group Group Immobilization per Ave. Length
(nominal) (meas.) (%) Adult (mm)
Control 0.0 µg/L 0 119 4.6
20 µg/L 11 µg/L 0 125 4.5
40 µg/L 15 µg/L 100 n/a n/a

The control daphnids released their first brood between days 7 and 10. The coefficient of variation (cv) for control fecundity was 16%, the guideline indicates that the cv for control reproduction in a “well run” study should be ≤25%. The test was considered acceptable as less than 20% of control organisms were immobilized and mean reproduction in the controls exceeded 60 young per adult.
Results with reference substance (positive control):
None used
Reported statistics and error estimates:
The chronic survival and reproduction NOEC for each test substance was based on empirical observation. The length NOEC was determined using the Bonferroni t-test using TOXSTAT software.
Validity criteria fulfilled:
yes
Conclusions:
The test substance showed no effects on survival, reproduction, or length at nominal and measured concentrations of 0.020 and 0.011mg/L, respectively. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) and No Observed Effect Concentration (NOEC) values are 0.020 and 0.011 mg/L, respectively, for these endpoints based upon nominal loading rates and measured (geometric means) concentrations.
Executive summary:

The test substance showed no effects on survival, reproduction, or length at nominal and measured concentrations of 0.020 and 0.011mg/L, respectively. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) and No Observed Effect Concentration (NOEC) values are 0.020 and 0.011 mg/L, respectively, for these endpoints based upon nominal loading rates and measured (geometric means) concentrations. The next highest loading rate and concentration tested, 0.040 and 0.015 mg/L, respectively, produced 100% immobilization of the adults exposed.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
February to March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were taken to confirm test material presence in test solutions and absence in control. Samples were taken from each test and control solution on Days 0, 7, 14, and 20 and the corresponding old solutions (three individual replicates) on Days 1, 8, 15 and 21, unless complete mortality occurred prior to the sampling event. Twenty-four samples from each group were analyzed during the twenty-one day study.
Vehicle:
no
Details on test solutions:
The exposure solutions were generated by aerating the test substance alone in a closed system using air stones at an air flow rate of 45 +/-5 cc/min. The vapor generated of the test substance from the aeration procedure flowed into aspirator bottles and bubbled through vehicle/dilution medium (reconstituted water). The test substance saturated vapor passed through air stones near the bottom of the aspirator bottles providing maximum contact between the test substance in the vapor phase and the vehicle/dilution medium. The systems were allowed to aerate/equilibrate for one day prior to preparing the exposure solutions for the first time. Ten replicates per treatment were tested.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test organisms were cultured at the test facility. The original culture was supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado, USA and received at the test facility on 11 April 2002.
For the test, eight adult daphnids were kept in 1-liter glass culture beakers with approximately 800 mL of reconstituted water (study vehicle/dilution water). The culture chamber was maintained at 20 ± 1 degrees C under a 16 hour light 8 hour dark photoperiod (10 - 20 foot/candles, 108 - 215 Lux). Day 0 cultures were started daily (at least five days per week) using eight <24 hour old neonates from culture beakers between 12 and 18 days old, exhibiting ≤20% adult mortality. Cultures were transferred to fresh reconstituted water on regular intervals to ensure that ≤24 hour old neonates are available for studies and to start new cultures.
Cultures of Daphnia magna were fed Pseudokirchneriella subcapitata (approximately 4.5 x 105 cells/mL) and 4.0 mL of a yeast / salmon starter / wheat grass (YTC) mixture per 800 mL daily (five days per week at a minimum). YTC and algae were supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado, USA.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
The hardness of the dilution water during the study was 150 mg/L as CaCO3.
Test temperature:
19.2 to 19.9 degrees C for test substance systems.
19.2 to 19.9 degrees C for control systems.
pH:
8.0 to 8.6 for test substance systems.
8.0 to 8.6 for control systems.
Dissolved oxygen:
7.8 to 9.2 mg/L for test substance systems.
7.9 to 9.1 mg/L for control systems.
Nominal and measured concentrations:
Nominal loading rates were control, 0.020, and 0.040 mg/L, which measured 0.0, 0.011, and 0.015 mg/L.
Details on test conditions:
Ten replicates per treatment were tested. One <24 hour old daphnid was added to each replicate. The adults were transferred to fresh solution daily. The test chambers were 125 mL capacity clear glass bottles containing approximately 125 mL of solution (no headspace). The test chambers were closed with foil lined screw type lids to minimize contamination, evaporation, and/or volatilization. A separate set of test chambers was used on a rotating basis for renewals. Feed was first added to each test chamber. Test chambers were filled with the appropriate amount of vehicle/dilution water. The adult Daphnia magna from the old test solution were transferred to the vehicle/dilution water using a glass pipette.
Diurnal light: approximately 16 hours light and 8 hours dark. Daylight intensity ranged from approximately 16 to 19 µE•m-2•s-1 during full daylight periods of the study.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.011 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
The test substance also showed no effects based on growth at 0.011 mg/L.
Details on results:
The following are the endpoint results for the control and test solutions:
Adult Neonates Adult
Group Group Immobilization per Ave. Length
(nominal) (meas.) (%) Adult (mm)
Control 0.0 µg/L 0 119 4.6
20 µg/L 11 µg/L 0 125 4.5
40 µg/L 15 µg/L 100 n/a n/a

The control daphnids released their first brood between days 7 and 10. The coefficient of variation (cv) for control fecundity was 16%, the guideline indicates that the cv for control reproduction in a “well run” study should be ≤25%. The test was considered acceptable as less than 20% of control organisms were immobilized and mean reproduction in the controls exceeded 60 young per adult.
Results with reference substance (positive control):
None used
Reported statistics and error estimates:
The chronic survival and reproduction NOEC for each test substance was based on empirical observation. The length NOEC was determined using the Bonferroni t-test using TOXSTAT software.
Validity criteria fulfilled:
yes
Conclusions:
The test substance showed no effects on survival, reproduction, or length at nominal and measured concentrations of 0.020 and 0.011mg/L, respectively. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) and No Observed Effect Concentration (NOEC) values are 0.020 and 0.011 mg/L, respectively, for these endpoints based upon nominal loading rates and measured (geometric means) concentrations.
Executive summary:

The test substance showed no effects on survival, reproduction, or length at nominal and measured concentrations of 0.020 and 0.011mg/L, respectively. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) and No Observed Effect Concentration (NOEC) values are 0.020 and 0.011 mg/L, respectively, for these endpoints based upon nominal loading rates and measured (geometric means) concentrations. The next highest loading rate and concentration tested, 0.040 and 0.015 mg/L, respectively, produced 100% immobilization of the adults exposed.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - September 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for analysis of new and old test solutions and controls were taken on days 1, 2, 3, 7, 10, 14, 17, and 21.
Vehicle:
no
Details on test solutions:
The water accommodated fraction (WAF) of the test material was prepared by stirring the test material in the exposure solution for approximately 2 days with a 1 to 2.5 hour settling period, after which the aqueous phase was removed and added to the test system. An equilibration study was conducted over a period of 96 hours prior to the definitive study to determine the approximate length of time needed to stir the test substance in the test media to acheive a saturated solution. Results suggested that a stirring period of approximately 2 days would be sufficient to reach equilibrium. The stirring period to develop WAFs used through the study ranged from 44.5 to 47 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
Organisms used in the test were from a laboratory culture were less than 24 hour old. The source of the culture was from Zeneca Brixham Laboratory from I.R.Ch.A., France.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
164 to 172 mg/L CaCO3
Test temperature:
19.9 to 20.5 degrees C
pH:
8.1 to 8.9
Dissolved oxygen:
6.3 to 9.1 mg/L
Nominal and measured concentrations:
The nominal treatment levels of the test substance included a control and 1000 mg/L, which measured <0.002 (the limit of detection) and 0.013 mg/L, respectively. The measured data represent the mean test substance concentration of samples taken from new and 24-hour old treatment solutions from days 1, 2, 3, 7, 10, 14, 17, and 21. The mean concentration of the test substance in new and old WAFs was 0.016 and 0.010 mg/L, respectively. The percent decrease in concentration of test material in solution from new to old WAFs on the sampling days ranged from 13 to 63%.
Details on test conditions:
The study was conducted in closed test systems that were renewed daily during the study. The test systems used were 150 ml glass Erlenmeyer flasks that were completely filled with media so as not to leave any headspace. The control and treatment level were each evaluated in 10 replicate test systems. One organism, less than 24 hours old, was added to each control and treatment solution flasks and were fed each day of the study. The feed was chlorella vulgaris at a rate of 0.3 mg carbon/daphnid/day. This rate was greater than that suggested in the OECD guideline (0.2 mg carbon/daphnid/day) because the test vessels were larger than specified by the guideline (50 to 100 ml). Light intensity was 485 lux.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Adult immobility: control - 0%; 1 mg/L - 10%
Total live young produced: control - 1453; 1 mg/L - 1122
Mean number of live young produced per surviving adult: control - 145; 1 mg/L - 124
Mean number of dead young produced per surviving adult: control - 0.2; 1 mg/L - 13

Reduction in fecundity in 1 mg/L treatment relative to control:
Brood 1 - 34%
Brood 2 - 28%
Brood 3 - 13%
Brood 4 - 3%
Brood 5 - 1.9%
Reported statistics and error estimates:
The significance of differences in the mean number of live young produced by adults in 21 days between the control and treatment solutions was assessed using a two-sample T-test.
Validity criteria fulfilled:
yes
Remarks:
The results show that survival of the adult control daphnids (100%) and the mean number of living offspring produced per surviving control parent (145) fulfilled the test validity criteria of >/= 80% and >/= 60 young/daphnia.
Conclusions:
During the 21-day study, the adult daphnids exposed to the test substance at a loading rate of 1 mg/L produced significantly fewer (<0.05) live offspring per adult than the adult daphnids in the control media. Therefore, the 21-day NOELR for reproduction was <1 mg/L. There was 10% immobility of parents in the treatment solution, therefore the EL50 can be reported as >1 mg/L.
Executive summary:

During the 21-day study, the adult daphnids exposed to the test substance at a loading rate of 1 mg/L produced significantly fewer (<0.05) live offspring per adult than the adult daphnids in the control media. Therefore, the 21-day NOELR for reproduction was <1 mg/L. There was also a greater number of dead offspring produced by the daphnids exposed to the test substance compared with those in the control. There was 10% immobility of parents in the treatment solution, therefore the EL50 can be reported as >1 mg/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
August - September 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for analysis of new and old test solutions and controls were taken on days 1, 2, 3, 7, 10, 14, 17, and 21.
Vehicle:
no
Details on test solutions:
The water accommodated fraction (WAF) of the test material was prepared by stirring the test material in the exposure solution for approximately 2 days with a 1 to 2.5 hour settling period, after which the aqueous phase was removed and added to the test system. An equilibration study was conducted over a period of 96 hours prior to the definitive study to determine the approximate length of time needed to stir the test substance in the test media to acheive a saturated solution. Results suggested that a stirring period of approximately 2 days would be sufficient to reach equilibrium. The stirring period to develop WAFs used through the study ranged from 44.5 to 47 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
Organisms used in the test were from a laboratory culture were less than 24 hour old. The source of the culture was from Zeneca Brixham Laboratory from I.R.Ch.A., France.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
164 to 172 mg/L CaCO3
Test temperature:
19.9 to 20.5 degrees C
pH:
8.1 to 8.9
Dissolved oxygen:
6.3 to 9.1 mg/L
Nominal and measured concentrations:
The nominal treatment levels of the test substance included a control and 1000 mg/L, which measured <0.002 (the limit of detection) and 0.013 mg/L, respectively. The measured data represent the mean test substance concentration of samples taken from new and 24-hour old treatment solutions from days 1, 2, 3, 7, 10, 14, 17, and 21. The mean concentration of the test substance in new and old WAFs was 0.016 and 0.010 mg/L, respectively. The percent decrease in concentration of test material in solution from new to old WAFs on the sampling days ranged from 13 to 63%.
Details on test conditions:
The study was conducted in closed test systems that were renewed daily during the study. The test systems used were 150 ml glass Erlenmeyer flasks that were completely filled with media so as not to leave any headspace. The control and treatment level were each evaluated in 10 replicate test systems. One organism, less than 24 hours old, was added to each control and treatment solution flasks and were fed each day of the study. The feed was chlorella vulgaris at a rate of 0.3 mg carbon/daphnid/day. This rate was greater than that suggested in the OECD guideline (0.2 mg carbon/daphnid/day) because the test vessels were larger than specified by the guideline (50 to 100 ml). Light intensity was 485 lux.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Adult immobility: control - 0%; 1 mg/L - 10%
Total live young produced: control - 1453; 1 mg/L - 1122
Mean number of live young produced per surviving adult: control - 145; 1 mg/L - 124
Mean number of dead young produced per surviving adult: control - 0.2; 1 mg/L - 13

Reduction in fecundity in 1 mg/L treatment relative to control:
Brood 1 - 34%
Brood 2 - 28%
Brood 3 - 13%
Brood 4 - 3%
Brood 5 - 1.9%
Reported statistics and error estimates:
The significance of differences in the mean number of live young produced by adults in 21 days between the control and treatment solutions was assessed using a two-sample T-test.
Validity criteria fulfilled:
yes
Remarks:
The results show that survival of the adult control daphnids (100%) and the mean number of living offspring produced per surviving control parent (145) fulfilled the test validity criteria of >/= 80% and >/= 60 young/daphnia.
Conclusions:
During the 21-day study, the adult daphnids exposed to the test substance at a loading rate of 1 mg/L produced significantly fewer (<0.05) live offspring per adult than the adult daphnids in the control media. Therefore, the 21-day NOELR for reproduction was <1 mg/L. There was 10% immobility of parents in the treatment solution, therefore the EL50 can be reported as >1 mg/L.
Executive summary:

During the 21-day study, the adult daphnids exposed to the test substance at a loading rate of 1 mg/L produced significantly fewer (<0.05) live offspring per adult than the adult daphnids in the control media. Therefore, the 21-day NOELR for reproduction was <1 mg/L. There was also a greater number of dead offspring produced by the daphnids exposed to the test substance compared with those in the control. There was 10% immobility of parents in the treatment solution, therefore the EL50 can be reported as >1 mg/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
No analytical measurements were taken. The low solubility of the test substance at the loading level tested was expected to result in exposure levels below the analytical detection limit for Total Organic Carbon (TOC) or Dissolved Organic Carbon (DOC).
Vehicle:
no
Details on test solutions:
Individual treatment solutions were prepared by adding the test substance, via syringe, to 4 L of laboratory blend water in glass aspirator bottles that were sealed with stoppers to minimize volatile loss of test material. The stock solutions were mixed for 24 hours. Mixing was performed using a magnetic stir plate and Teflon stir bar. After mixing, the solutions were allowed to settle for one hour at test temperature. The Water Accommodated Fraction (WAF) was then removed through the outlet at the bottom of the mixing vessel and added to the test chambers.
Test organisms (species):
Daphnia magna
Details on test organisms:
The Daphnia were cultured in-house, were <24 hours old, and were from 17-day old parents. Test organisms were fed daily when solutions were renewed with Pseudokirchneriella subcapitata to provide approximately 4.7 x 10E5 cells/mL. The daphnia were fed a carbon equivalent of approximately 0.5 mg C per adult per day which is purposely higher than the guideline to achieve an acceptable level of neonate production.

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
The hardness of the dilution water during the study was 114-120 mg/L as CaCO3.
Test temperature:
Mean test temperature was 20.6 degrees C (S.D. = 0.3) for test substance and control systems.
pH:
7.0 to 7.9 for test substance and control systems.
Dissolved oxygen:
7.3 to 8.7 mg/L for test substance and control systems.
Nominal and measured concentrations:
Nominal loading rates were control and 1 mg/L. Analytical measurements were not taken.
Details on test conditions:
The test chambers were 55 mL capacity glass Erlenmeyer flasks with ground glass stoppers (no headspace). One daphnid was added to each of 10 replicates. The test was performed using a static daily renewal of exposure solutions. Observations for abnormal or immobilized daphnids and neonates were made on each replicate at approximately 24-hour intervals. Diurnal light: approximately 17 hours light and 7 hours dark. Water Quality was measured twice per week.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
The test substance also showed no effects based on growth.
Details on results:
The following are the endpoint results for the control and test solution:
Adult Neonates
Group Immobilization per
(%) Adult
Control 20 61
1 mg/L 10 47

The control daphnids released their first brood between days 7 and 9. The coefficient of variation (cv) for control fecundity was 33%. The test was considered acceptable as control organisms immobilization did not exceed 20% and mean reproduction in the controls exceeded 60 young per adult.
Results with reference substance (positive control):
None used
Reported statistics and error estimates:
Statistical analyses were conducted using the T-test for reproduction from ToxStat 3.4 (West Inc. 1994).
Validity criteria fulfilled:
yes
Conclusions:
The test substance showed no effects on survival, reproduction, or length at a nominal loading of 1 mg/L. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) = 1.0 mg/L for these endpoints based upon nominal loading levels.
Executive summary:

The test substance showed no effects on survival, reproduction, or length at a nominal loading of 1 mg/L. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) = 1.0 mg/L for these endpoints based upon nominal loading level.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
No analytical measurements were taken. The low solubility of the test substance at the loading level tested was expected to result in exposure levels below the analytical detection limit for Total Organic Carbon (TOC) or Dissolved Organic Carbon (DOC).
Vehicle:
no
Details on test solutions:
Individual treatment solutions were prepared by adding the test substance, via syringe, to 4 L of laboratory blend water in glass aspirator bottles that were sealed with stoppers to minimize volatile loss of test material. The stock solutions were mixed for 24 hours. Mixing was performed using a magnetic stir plate and Teflon stir bar. After mixing, the solutions were allowed to settle for one hour at test temperature. The Water Accommodated Fraction (WAF) was then removed through the outlet at the bottom of the mixing vessel and added to the test chambers.
Test organisms (species):
Daphnia magna
Details on test organisms:
The Daphnia were cultured in-house, were <24 hours old, and were from 17-day old parents. Test organisms were fed daily when solutions were renewed with Pseudokirchneriella subcapitata to provide approximately 4.7 x 10E5 cells/mL. The daphnia were fed a carbon equivalent of approximately 0.5 mg C per adult per day which is purposely higher than the guideline to achieve an acceptable level of neonate production.

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
The hardness of the dilution water during the study was 114-120 mg/L as CaCO3.
Test temperature:
Mean test temperature was 20.6 degrees C (S.D. = 0.3) for test substance and control systems.
pH:
7.0 to 7.9 for test substance and control systems.
Dissolved oxygen:
7.3 to 8.7 mg/L for test substance and control systems.
Nominal and measured concentrations:
Nominal loading rates were control and 1 mg/L. Analytical measurements were not taken.
Details on test conditions:
The test chambers were 55 mL capacity glass Erlenmeyer flasks with ground glass stoppers (no headspace). One daphnid was added to each of 10 replicates. The test was performed using a static daily renewal of exposure solutions. Observations for abnormal or immobilized daphnids and neonates were made on each replicate at approximately 24-hour intervals. Diurnal light: approximately 17 hours light and 7 hours dark. Water Quality was measured twice per week.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
The test substance also showed no effects based on growth.
Details on results:
The following are the endpoint results for the control and test solution:
Adult Neonates
Group Immobilization per
(%) Adult
Control 20 61
1 mg/L 10 47

The control daphnids released their first brood between days 7 and 9. The coefficient of variation (cv) for control fecundity was 33%. The test was considered acceptable as control organisms immobilization did not exceed 20% and mean reproduction in the controls exceeded 60 young per adult.
Results with reference substance (positive control):
None used
Reported statistics and error estimates:
Statistical analyses were conducted using the T-test for reproduction from ToxStat 3.4 (West Inc. 1994).
Validity criteria fulfilled:
yes
Conclusions:
The test substance showed no effects on survival, reproduction, or length at a nominal loading of 1 mg/L. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) = 1.0 mg/L for these endpoints based upon nominal loading levels.
Executive summary:

The test substance showed no effects on survival, reproduction, or length at a nominal loading of 1 mg/L. Therefore, the 21-Day No Observed Effect Loading Rate (NOELR) = 1.0 mg/L for these endpoints based upon nominal loading level.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2001-07-24 to 2001-08-1
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study. However, information on the substance is lacking, particularly the density of the susbtance would have been necessary to be able to calculate the end-point values as concentrations (mg/L for instance) instead of % WAF as it is in the study report. MSDS needed.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: EPA 1002.0: Ceriodaphnia survival and reproduction test
Deviations:
no
Principles of method if other than guideline:
Ceriodaphnia dubia are exposed to the substance and the test is ended when 60% of the surviving control females produced 3 broods of young.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No test surrogate or analogue material
Analytical monitoring:
no
Details on sampling:
Not applicable (no analytical monitoring)
Vehicle:
no
Details on test solutions:
Water accommodated fractions (WAFs) were prepraed on alternate days durinfg testing accoding to methods described by Anderson et al. (Anderson, 1974). Ten parts of the test water and one part of the test material were combined to prepare each WAF; actual volumes of liquids were dependent on the volume needed for testing. The solutions were mixed for 20 +/- 1 hours using a magnetic stir bar and a stir plate. The vortex produced during mixing was not allowed to exceed 25% of the distance from the liquid surface to the bottom of the mixing chamber. After mixing, the WAFs were extracted from a point near the bottom of the mixing chambers by siphoning.
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: obtained from in-house cultures at the Fort Collins Environmental Toxicology Laboratory
- Age: At the time of test initiation, the test organisms were less than 24-hour old
- Feeding during test
- No feeding during the range-finding test
- Feeding during the definitive test:
- Food type: yeast-trout chow-cereal leaves/algae mix
- Amount: 0.2 ml per test chamber
- Frequency: daily


ACCLIMATION
- Acclimation period: 24 h after hatching
- Acclimation conditions: same as test
- Health during acclimation (any mortality observed): Good physical condition
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
6 d
Post exposure observation period:
Data were gathered through 7 days, although 60% of the surviving control females had third brood by day 6, when the test should have been terminated.
Hardness:
96-106 mg/L CaCO3
Test temperature:
20+/- 1°C
pH:
8.0-8.3
Dissolved oxygen:
>=6.9 mg/L
Salinity:
Conductivity: 339-346 µS/cm
Nominal and measured concentrations:
0, 12.5, 25, 50, 75 and 100% WAF
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 30 mL plastic beakers with 15 mL test medium
- No aeration during any of the tests
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates):10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted moderatly hard water (USEPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. EPA/600/4-91/002)
- Chlorine <0.05 mg/L
- Alkalinity: 66-69 mg/L CaCO3
- Conductivity:339-364 µS/cm
- Intervals of water quality measurement: daily


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16 h L / 8 h D (fluorescent lighting)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality of adults (defined as no visible movement after gentle prodding with a blunt probe
- Total number of young produced per adult


RANGE-FINDING STUDY
- Test concentrations: 0, 1, 10 and 100% WAF for 24 hours
- Results used to determine the conditions for the definitive study: the high concentration for the definitive test is set at 100% WAF.
Reference substance (positive control):
yes
Remarks:
Sodium chloride NaCl
Key result
Duration:
6 d
Dose descriptor:
EL50
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Not reached
Duration:
6 d
Dose descriptor:
LOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No statistical differences between control and treated
Key result
Duration:
6 d
Dose descriptor:
NOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No statistical differences between control and treated
Key result
Duration:
6 d
Dose descriptor:
EL50
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Not reached
Duration:
6 d
Dose descriptor:
LOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: No statistical differences between control and treated
Key result
Duration:
6 d
Dose descriptor:
NOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: No statistical differences between control and treated
Details on results:
- Mortality of parent animals: see table 1 (§ Remarks on results including tables and figures)
- No. of offspring produced per day per female: see table 1 (§ Remarks on results including tables and figures)
Results with reference substance (positive control):
IC25 = 554 mg/L (95% Confidence interval: 136-745) (NaCl as mg/L Cl-)
Reported statistics and error estimates:
See table 2 (§ Remarks on results including tables and figures)

Table 1. Chronic exposure ofCeriodaphnia dubiato Vista C13-C16: Definitve test results

 

Test concentration (% WAF)

Survival (%) on each test day

 

Mean young per female

1

2

3

4

5

6

0 (Control)

100

100

100

100

90

90

18.0

12.5

100

100

100

100

100

100

16.8

25

100

100

100

90

90

90

15.7

50

100

100

100

100

90

90

13.0

75

100

100

100

100

100

90

15.8

100

100

100

100

100

100

100

10.6

 

 

Table 2. Chronic exposure ofCeriodaphnia dubiato Vista C13-C16: Statistical methods

 

End point

Comparison

Procedure

Survival

Significant reduction relative to the dilution water control

Inspection

Treshold inhibition concentration (IC25/EL25)

Inspection

Reproduction

Normality

Chi-Square test (α=0.01)

Homogeneity of variance

Bartlett’s test (α=0.01)

Significant reduction relative to the dilution water control

Dunnett’s test (α=0.05)

Treshold inhibition concentration (IC25/EL25)

Linear interpolation

Validity criteria fulfilled:
yes
Conclusions:
The NOELR for Ceriodaphnia dubia exposed to Vista C13-C16 was >100% WAF based on mortality and reproduction (no statistical differences between treatment groups)
Executive summary:

The study was performed to evaluate the toxicity of the water accommodated fractions (WAFs) of the test substance Vista C13-C16 to the water fleaCeriodaphnia dubia, in a semi-static system. The test was ended when 60% of the control females had produced 3 broods (6 days).

The water accommodated fractions were prepared by mixing 1 volume of the test substance with 10 volumes of dilution water. The dilution water was reconstituted moderately hard water prepared in the laboratory according to USEPA (USEPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. EPA/600/4-91/002). A range-finding test with 0, 1, 10 and 100 % WAF allowed setting the high concentration at 100% WAF for the definitive test. The definitive test was performed with 0, 12.5, 25, 50, 75 and 100 % WAF. The stock solution was prepared on alternate days as WAF by mixing 1 volume of test substance to 10 volumes of dilution water. The solution was mixed for 20±1 hours and the WAF was extracted from a point near the bottom of the mixing chamber by siphoning.

10 replicates, each containing one organism, were tested for each treatment. The test media were renewed daily. Mortality and reproduction were assessed daily.

 

An EL25(reported as IC25in the study report) was calculated for reproduction by linear interpolation:

EL25= 80.9 % WAF

 

However, the statistical study showed that there was no statistical difference between treatment groups (including control group) for mortality as well as for reproduction. Therefore, the No Observed Effect Loading Rate based on adult mortality and reproduction (expressed as number of young per female) was set at > 100 % WAF.

NOELR = 100% WAF

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
from 2001-07-24 to 2001-08-1
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study. However, information on the substance is lacking, particularly the density of the susbtance would have been necessary to be able to calculate the end-point values as concentrations (mg/L for instance) instead of % WAF as it is in the study report. MSDS needed.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: EPA 1002.0: Ceriodaphnia survival and reproduction test
Deviations:
no
Principles of method if other than guideline:
Ceriodaphnia dubia are exposed to the substance and the test is ended when 60% of the surviving control females produced 3 broods of young.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No test surrogate or analogue material
Analytical monitoring:
no
Details on sampling:
Not applicable (no analytical monitoring)
Vehicle:
no
Details on test solutions:
Water accommodated fractions (WAFs) were prepraed on alternate days durinfg testing accoding to methods described by Anderson et al. (Anderson, 1974). Ten parts of the test water and one part of the test material were combined to prepare each WAF; actual volumes of liquids were dependent on the volume needed for testing. The solutions were mixed for 20 +/- 1 hours using a magnetic stir bar and a stir plate. The vortex produced during mixing was not allowed to exceed 25% of the distance from the liquid surface to the bottom of the mixing chamber. After mixing, the WAFs were extracted from a point near the bottom of the mixing chambers by siphoning.
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: obtained from in-house cultures at the Fort Collins Environmental Toxicology Laboratory
- Age: At the time of test initiation, the test organisms were less than 24-hour old
- Feeding during test
- No feeding during the range-finding test
- Feeding during the definitive test:
- Food type: yeast-trout chow-cereal leaves/algae mix
- Amount: 0.2 ml per test chamber
- Frequency: daily


ACCLIMATION
- Acclimation period: 24 h after hatching
- Acclimation conditions: same as test
- Health during acclimation (any mortality observed): Good physical condition
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
6 d
Post exposure observation period:
Data were gathered through 7 days, although 60% of the surviving control females had third brood by day 6, when the test should have been terminated.
Hardness:
96-106 mg/L CaCO3
Test temperature:
20+/- 1°C
pH:
8.0-8.3
Dissolved oxygen:
>=6.9 mg/L
Salinity:
Conductivity: 339-346 µS/cm
Nominal and measured concentrations:
0, 12.5, 25, 50, 75 and 100% WAF
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 30 mL plastic beakers with 15 mL test medium
- No aeration during any of the tests
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates):10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted moderatly hard water (USEPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. EPA/600/4-91/002)
- Chlorine <0.05 mg/L
- Alkalinity: 66-69 mg/L CaCO3
- Conductivity:339-364 µS/cm
- Intervals of water quality measurement: daily


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16 h L / 8 h D (fluorescent lighting)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality of adults (defined as no visible movement after gentle prodding with a blunt probe
- Total number of young produced per adult


RANGE-FINDING STUDY
- Test concentrations: 0, 1, 10 and 100% WAF for 24 hours
- Results used to determine the conditions for the definitive study: the high concentration for the definitive test is set at 100% WAF.
Reference substance (positive control):
yes
Remarks:
Sodium chloride NaCl
Key result
Duration:
6 d
Dose descriptor:
EL50
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Not reached
Duration:
6 d
Dose descriptor:
LOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No statistical differences between control and treated
Key result
Duration:
6 d
Dose descriptor:
NOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No statistical differences between control and treated
Key result
Duration:
6 d
Dose descriptor:
EL50
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Not reached
Duration:
6 d
Dose descriptor:
LOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: No statistical differences between control and treated
Key result
Duration:
6 d
Dose descriptor:
NOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: No statistical differences between control and treated
Details on results:
- Mortality of parent animals: see table 1 (§ Remarks on results including tables and figures)
- No. of offspring produced per day per female: see table 1 (§ Remarks on results including tables and figures)
Results with reference substance (positive control):
IC25 = 554 mg/L (95% Confidence interval: 136-745) (NaCl as mg/L Cl-)
Reported statistics and error estimates:
See table 2 (§ Remarks on results including tables and figures)

Table 1. Chronic exposure ofCeriodaphnia dubiato Vista C13-C16: Definitve test results

 

Test concentration (% WAF)

Survival (%) on each test day

 

Mean young per female

1

2

3

4

5

6

0 (Control)

100

100

100

100

90

90

18.0

12.5

100

100

100

100

100

100

16.8

25

100

100

100

90

90

90

15.7

50

100

100

100

100

90

90

13.0

75

100

100

100

100

100

90

15.8

100

100

100

100

100

100

100

10.6

 

 

Table 2. Chronic exposure ofCeriodaphnia dubiato Vista C13-C16: Statistical methods

 

End point

Comparison

Procedure

Survival

Significant reduction relative to the dilution water control

Inspection

Treshold inhibition concentration (IC25/EL25)

Inspection

Reproduction

Normality

Chi-Square test (α=0.01)

Homogeneity of variance

Bartlett’s test (α=0.01)

Significant reduction relative to the dilution water control

Dunnett’s test (α=0.05)

Treshold inhibition concentration (IC25/EL25)

Linear interpolation

Validity criteria fulfilled:
yes
Conclusions:
The NOELR for Ceriodaphnia dubia exposed to Vista C13-C16 was >100% WAF based on mortality and reproduction (no statistical differences between treatment groups)
Executive summary:

The study was performed to evaluate the toxicity of the water accommodated fractions (WAFs) of the test substance Vista C13-C16 to the water fleaCeriodaphnia dubia, in a semi-static system. The test was ended when 60% of the control females had produced 3 broods (6 days).

The water accommodated fractions were prepared by mixing 1 volume of the test substance with 10 volumes of dilution water. The dilution water was reconstituted moderately hard water prepared in the laboratory according to USEPA (USEPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. EPA/600/4-91/002). A range-finding test with 0, 1, 10 and 100 % WAF allowed setting the high concentration at 100% WAF for the definitive test. The definitive test was performed with 0, 12.5, 25, 50, 75 and 100 % WAF. The stock solution was prepared on alternate days as WAF by mixing 1 volume of test substance to 10 volumes of dilution water. The solution was mixed for 20±1 hours and the WAF was extracted from a point near the bottom of the mixing chamber by siphoning.

10 replicates, each containing one organism, were tested for each treatment. The test media were renewed daily. Mortality and reproduction were assessed daily.

 

An EL25(reported as IC25in the study report) was calculated for reproduction by linear interpolation:

EL25= 80.9 % WAF

 

However, the statistical study showed that there was no statistical difference between treatment groups (including control group) for mortality as well as for reproduction. Therefore, the No Observed Effect Loading Rate based on adult mortality and reproduction (expressed as number of young per female) was set at > 100 % WAF.

NOELR = 100% WAF

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 2001-07-24 to 2001-08-1
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study. However, end-point values are expressed as % WAF. An attempt was then made to evaluate the end-point values as mg/L from the density of the substance. MSDS provided by the manufacturer.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: EPA 1002.0: Ceriodaphnia survival and reproduction test
Deviations:
no
Principles of method if other than guideline:
Ceriodaphnia dubia are exposed to the substance and the test is ended when 60% of the surviving control females produced 3 broods of young.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No test surrogate or analogue material
Analytical monitoring:
no
Details on sampling:
Not applicable (no analytical monitoring)
Vehicle:
no
Details on test solutions:
Water accommodated fractions (WAFs) were prepraed on alternate days durinfg testing accoding to methods described by Anderson et al. (Anderson, 1974). Ten parts of the test water and one part of the test material were combined to prepare each WAF; actual volumes of liquids were dependent on the volume needed for testing. The solutions were mixed for 20 +/- 1 hours using a magnetic stir bar and a stir plate. The vortex produced during mixing was not allowed to exceed 25% of the distance from the liquid surface to the bottom of the mixing chamber. After mixing, the WAFs were extracted from a point near the bottom of the mixing chambers by siphoning.
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: obtained from in-house cultures at the Fort Collins Environmental Toxicology Laboratory
- Age: At the time of test initiation, the test organisms were less than 24-hour old
- Feeding during test
- No feeding during the range-finding test
- Feeding during the definitive test:
- Food type: yeast-trout chow-cereal leaves/algae mix
- Amount: 0.2 ml per test chamber
- Frequency: daily


ACCLIMATION
- Acclimation period: 24 h after hatching
- Acclimation conditions: same as test
- Health during acclimation (any mortality observed): Good physical condition
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
8 d
Post exposure observation period:
No post-exposure observation.
Hardness:
96-100 mg/L CaCO3
Test temperature:
20+/- 1°C
pH:
8.0-8.3
Dissolved oxygen:
>=6.9 mg/L
Salinity:
Conductivity: 340-351 µS/cm
Nominal and measured concentrations:
0, 12.5, 25, 50, 75 and 100% WAF corresponding to approximately 0, 8.75, 17.5, 35, 52.5 and 70 mg/L (nominal loadings as calculated from density value)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 30 mL plastic beakers with 15 mL test medium
- No aeration during any of the tests
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates):10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted moderatly hard water (USEPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. EPA/600/4-91/002)
- Chlorine <0.05 mg/L
- Alkalinity: 66-69 mg/L CaCO3
- Conductivity:339-364 µS/cm
- Intervals of water quality measurement: daily


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16 h L / 8 h D (fluorescent lighting)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality of adults (defined as no visible movement after gentle prodding with a blunt probe
- Total number of young produced per adult


RANGE-FINDING STUDY
- Test concentrations: 0, 1, 10 and 100% WAF for 24 hours (i.e. nominal loadings: 0, 0.7, 7 and 70 mg/L approximately, as calculated from the density value)
- Results used to determine the conditions for the definitive study: the high concentration for the definitive test is set at 100% WAF.
Reference substance (positive control):
yes
Remarks:
Sodium chloride NaCl
Key result
Duration:
8 d
Dose descriptor:
EL50
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Duration:
8 d
Dose descriptor:
LOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Key result
Duration:
8 d
Dose descriptor:
NOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Key result
Duration:
8 d
Dose descriptor:
EL50
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Duration:
8 d
Dose descriptor:
LOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Key result
Duration:
8 d
Dose descriptor:
NOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Details on results:
- Mortality of parent animals: see table 1 (§ Remarks on results including tables and figures)
- No. of offspring produced per day per female: see table 1 (§ Remarks on results including tables and figures)
Results with reference substance (positive control):
IC25 = 554 mg/L (95% Confidence interval: 136-745) (NaCl as mg/L Cl-)
Reported statistics and error estimates:
See table 2 (§ Remarks on results including tables and figures)

Table 1. Chronic exposure ofCeriodaphnia dubiato Linpar 1416-V: Definitve test results

 

Concentration (% WAF)

Survival (%) on each test day

Mean young per female

1

2

3

4

5

6

7

8

0 (Control)

100

100

100

100

100

100

90

90

29.7

12.5

100

100

100

100

100

100

100

100

34.2

25

100

100

100

100

100

100

100

100

13.5

50

100

100

100

100

90

90

90

90

23.6

75

100

100

100

90

100

100

90

90

25.3

100

100

100

100

100

100

100

100

100

19.9

 

 

Table 2. Chronic exposure ofCeriodaphnia dubiato Linpar 1416-V: Statistical methods

 

End point

Comparison

Procedure

Survival

Significant reduction relative to the dilution water control

Inspection

Treshold inhibition concentration (IC25/EL25)

Inspection

Reproduction

Normality

Chi-Square test (α=0.01)

Homogeneity of variance

Bartlett’s test (α=0.01)

Significant reduction relative to the dilution water control

Dunnett’s test (α=0.05)

Treshold inhibition concentration (IC25/EL25)

Linear interpolation

Validity criteria fulfilled:
yes
Conclusions:
The NOELR for Ceriodaphnia dubia exposed to Linpar 1416-V was >100% WAF based on mortality and reproduction (no statistical differences between treatment groups)
Executive summary:

The study was performed to evaluate the toxicity of the water accommodated fractions (WAFs) of the test substance Linpar 1416-V to the water fleaCeriodaphnia dubia, in a semi-static system. The test was ended when 60% of the control females had produced 3 broods (8 days).

The water accommodated fractions were prepared by mixing 1 volume of the test substance with 10 volumes of dilution water. The dilution water was reconstituted moderately hard water prepared in the laboratory according to USEPA (USEPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. EPA/600/4-91/002). A range-finding test with 0, 1, 10 and 100 % WAF (corresponding approximately to 0, 0.7, 7 and 70 mg/L nominal loading according to substance density) allowed setting the high concentration at 100% WAF (i.e. approx. 70 mg/L nominal loading) for the definitive test. The definitive test was performed with 0, 12.5, 25, 50, 75 and 100 % WAF (corresponding to approx. 0, 8.75, 17.5, 35, 52.5 and 70 mg/L nominal laoding according to substance density). The stock solution was prepared on alternate days as WAF by mixing 1 volume of test substance to 10 volumes of dilution water. The solution was mixed for 20±1 hours and the WAF was extracted from a point near the bottom of the mixing chamber by siphoning.

10 replicates, each containing one organism, were tested for each treatment. The test media were renewed daily. Mortality and reproduction were assessed daily.

 

An EL25(reported as IC25in the study report) was calculated for reproduction by linear interpolation:

EL25= 77.7 % WAF (approx. 54 mg/L nominal loading)

 

However, the statistical study showed that there was no statistical difference between treatment groups (including control group) for mortality as well as for reproduction. Therefore, the No Observed Effect Loading Rate based on adult mortality and reproduction (expressed as number of young per female) was set at > 100 % WAF (i.e. approx. > 70 mg/L nominal loading according to substance density).

 

NOELR > 100 % WAF (i.e. > approx. 70 mg/L nominal loading)

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
from 2001-07-24 to 2001-08-1
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study. However, end-point values are expressed as % WAF. An attempt was then made to evaluate the end-point values as mg/L from the density of the substance. MSDS provided by the manufacturer.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: EPA 1002.0: Ceriodaphnia survival and reproduction test
Deviations:
no
Principles of method if other than guideline:
Ceriodaphnia dubia are exposed to the substance and the test is ended when 60% of the surviving control females produced 3 broods of young.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No test surrogate or analogue material
Analytical monitoring:
no
Details on sampling:
Not applicable (no analytical monitoring)
Vehicle:
no
Details on test solutions:
Water accommodated fractions (WAFs) were prepraed on alternate days durinfg testing accoding to methods described by Anderson et al. (Anderson, 1974). Ten parts of the test water and one part of the test material were combined to prepare each WAF; actual volumes of liquids were dependent on the volume needed for testing. The solutions were mixed for 20 +/- 1 hours using a magnetic stir bar and a stir plate. The vortex produced during mixing was not allowed to exceed 25% of the distance from the liquid surface to the bottom of the mixing chamber. After mixing, the WAFs were extracted from a point near the bottom of the mixing chambers by siphoning.
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: obtained from in-house cultures at the Fort Collins Environmental Toxicology Laboratory
- Age: At the time of test initiation, the test organisms were less than 24-hour old
- Feeding during test
- No feeding during the range-finding test
- Feeding during the definitive test:
- Food type: yeast-trout chow-cereal leaves/algae mix
- Amount: 0.2 ml per test chamber
- Frequency: daily


ACCLIMATION
- Acclimation period: 24 h after hatching
- Acclimation conditions: same as test
- Health during acclimation (any mortality observed): Good physical condition
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
8 d
Post exposure observation period:
No post-exposure observation.
Hardness:
96-100 mg/L CaCO3
Test temperature:
20+/- 1°C
pH:
8.0-8.3
Dissolved oxygen:
>=6.9 mg/L
Salinity:
Conductivity: 340-351 µS/cm
Nominal and measured concentrations:
0, 12.5, 25, 50, 75 and 100% WAF corresponding to approximately 0, 8.75, 17.5, 35, 52.5 and 70 mg/L (nominal loadings as calculated from density value)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 30 mL plastic beakers with 15 mL test medium
- No aeration during any of the tests
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates):10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted moderatly hard water (USEPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. EPA/600/4-91/002)
- Chlorine <0.05 mg/L
- Alkalinity: 66-69 mg/L CaCO3
- Conductivity:339-364 µS/cm
- Intervals of water quality measurement: daily


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16 h L / 8 h D (fluorescent lighting)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality of adults (defined as no visible movement after gentle prodding with a blunt probe
- Total number of young produced per adult


RANGE-FINDING STUDY
- Test concentrations: 0, 1, 10 and 100% WAF for 24 hours (i.e. nominal loadings: 0, 0.7, 7 and 70 mg/L approximately, as calculated from the density value)
- Results used to determine the conditions for the definitive study: the high concentration for the definitive test is set at 100% WAF.
Reference substance (positive control):
yes
Remarks:
Sodium chloride NaCl
Key result
Duration:
8 d
Dose descriptor:
EL50
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Duration:
8 d
Dose descriptor:
LOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Key result
Duration:
8 d
Dose descriptor:
NOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Key result
Duration:
8 d
Dose descriptor:
EL50
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Duration:
8 d
Dose descriptor:
LOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Key result
Duration:
8 d
Dose descriptor:
NOELR
Effect conc.:
> 100 other: % WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (> approx. 70 mg/L) - No statistical differences between treatment groups
Details on results:
- Mortality of parent animals: see table 1 (§ Remarks on results including tables and figures)
- No. of offspring produced per day per female: see table 1 (§ Remarks on results including tables and figures)
Results with reference substance (positive control):
IC25 = 554 mg/L (95% Confidence interval: 136-745) (NaCl as mg/L Cl-)
Reported statistics and error estimates:
See table 2 (§ Remarks on results including tables and figures)

Table 1. Chronic exposure ofCeriodaphnia dubiato Linpar 1416-V: Definitve test results

 

Concentration (% WAF)

Survival (%) on each test day

Mean young per female

1

2

3

4

5

6

7

8

0 (Control)

100

100

100

100

100

100

90

90

29.7

12.5

100

100

100

100

100

100

100

100

34.2

25

100

100

100

100

100

100

100

100

13.5

50

100

100

100

100

90

90

90

90

23.6

75

100

100

100

90

100

100

90

90

25.3

100

100

100

100

100

100

100

100

100

19.9

 

 

Table 2. Chronic exposure ofCeriodaphnia dubiato Linpar 1416-V: Statistical methods

 

End point

Comparison

Procedure

Survival

Significant reduction relative to the dilution water control

Inspection

Treshold inhibition concentration (IC25/EL25)

Inspection

Reproduction

Normality

Chi-Square test (α=0.01)

Homogeneity of variance

Bartlett’s test (α=0.01)

Significant reduction relative to the dilution water control

Dunnett’s test (α=0.05)

Treshold inhibition concentration (IC25/EL25)

Linear interpolation

Validity criteria fulfilled:
yes
Conclusions:
The NOELR for Ceriodaphnia dubia exposed to Linpar 1416-V was >100% WAF based on mortality and reproduction (no statistical differences between treatment groups)
Executive summary:

The study was performed to evaluate the toxicity of the water accommodated fractions (WAFs) of the test substance Linpar 1416-V to the water fleaCeriodaphnia dubia, in a semi-static system. The test was ended when 60% of the control females had produced 3 broods (8 days).

The water accommodated fractions were prepared by mixing 1 volume of the test substance with 10 volumes of dilution water. The dilution water was reconstituted moderately hard water prepared in the laboratory according to USEPA (USEPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. EPA/600/4-91/002). A range-finding test with 0, 1, 10 and 100 % WAF (corresponding approximately to 0, 0.7, 7 and 70 mg/L nominal loading according to substance density) allowed setting the high concentration at 100% WAF (i.e. approx. 70 mg/L nominal loading) for the definitive test. The definitive test was performed with 0, 12.5, 25, 50, 75 and 100 % WAF (corresponding to approx. 0, 8.75, 17.5, 35, 52.5 and 70 mg/L nominal laoding according to substance density). The stock solution was prepared on alternate days as WAF by mixing 1 volume of test substance to 10 volumes of dilution water. The solution was mixed for 20±1 hours and the WAF was extracted from a point near the bottom of the mixing chamber by siphoning.

10 replicates, each containing one organism, were tested for each treatment. The test media were renewed daily. Mortality and reproduction were assessed daily.

 

An EL25(reported as IC25in the study report) was calculated for reproduction by linear interpolation:

EL25= 77.7 % WAF (approx. 54 mg/L nominal loading)

 

However, the statistical study showed that there was no statistical difference between treatment groups (including control group) for mortality as well as for reproduction. Therefore, the No Observed Effect Loading Rate based on adult mortality and reproduction (expressed as number of young per female) was set at > 100 % WAF (i.e. approx. > 70 mg/L nominal loading according to substance density).

 

NOELR > 100 % WAF (i.e. > approx. 70 mg/L nominal loading)

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
(Q)SAR
Adequacy of study:
key study
Study period:
Not applicable, calculated value
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This summary has a reliability of 2 because the results are estimated using a computer model that is appropriate for use with this hydrocarbon substance.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aquatic toxicity was estimated by a QSAR, the Petrotox computer model. This model combines a partitioning model used to calculate the aqueous concentration of hydrocarbon components with the Target Lipid Model used to calculate acute and chronic toxicity of non-polar narcotic chemicals. Petrotox computes toxicity based on the summation of the aqueous-phase concentrations of hydrocarbon block(s) that represent a hydrocarbon substance and membrane-water partition coefficients (KMW) that describe the partitioning of the hydrocarbons between the water and organism.
GLP compliance:
no
Remarks:
The data were calculated by a computer model.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No test surrogate nor analogue material
Analytical monitoring:
not required
Details on sampling:
Not applicable (calculated data)
Vehicle:
no
Details on test solutions:
Not applicable (calculated data)
Test organisms (species):
Daphnia magna
Details on test organisms:
Not applicable (calculated data)
Test type:
other: QSAR modeled data
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
Not applicable (calculated data)
Hardness:
Not applicable (calculated data)
Test temperature:
Not applicable (calculated data)
pH:
Not applicable (calculated data)
Dissolved oxygen:
Not applicable (calculated data)
Salinity:
Not applicable (calculated data)
Nominal and measured concentrations:
Not applicable (calculated data)
Details on test conditions:
Not applicable (calculated data)
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
No details (calculated data) - See inforamtion on substance composition in "Overall Remarks" section
Results with reference substance (positive control):
No reference substance required
Reported statistics and error estimates:
Not applicable

No other information

Validity criteria fulfilled:
yes
Conclusions:
Results of computer modelling to estimate chronicity in a 21-day freshwater invertebrate study, based on reproduction, show that this substance will not produce toxicity at or below its maximum attainable water solubility
Executive summary:

The aquatic toxicity of was estimated using the Petrotox computer model, which combines a partitioning model used to calculate the aqueous concentration of hydrocarbon components as a function of substance loading with the Target Lipid Model used to calculate acute and chronic toxicity of non-polar narcotic chemicals. Petrotox computes toxicity based on the summation of the aqueous-phase concentrations of hydrocarbon block(s) that represent a hydrocarbon substance and membrane-water partitioning coefficients (KMW) that describe the partitioning of the hydrocarbons between the water and organism. Results of computer modelling to estimate chronicity in a 21-day freshwater invertebrate study, based on reproduction, show that this substance will not produce toxicity at or below its maximum attainable water solubility.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not applicable, calculated value
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This summary has a reliability of 2 because the results are estimated using a computer model that is appropriate for use with this hydrocarbon substance.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aquatic toxicity was estimated by a QSAR, the Petrotox computer model. This model combines a partitioning model used to calculate the aqueous concentration of hydrocarbon components with the Target Lipid Model used to calculate acute and chronic toxicity of non-polar narcotic chemicals. Petrotox computes toxicity based on the summation of the aqueous-phase concentrations of hydrocarbon block(s) that represent a hydrocarbon substance and membrane-water partition coefficients (KMW) that describe the partitioning of the hydrocarbons between the water and organism.
GLP compliance:
no
Remarks:
The data were calculated by a computer model.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No test surrogate nor analogue material
Analytical monitoring:
not required
Details on sampling:
Not applicable (calculated data)
Vehicle:
no
Details on test solutions:
Not applicable (calculated data)
Test organisms (species):
Daphnia magna
Details on test organisms:
Not applicable (calculated data)
Test type:
other: QSAR modeled data
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
Not applicable (calculated data)
Hardness:
Not applicable (calculated data)
Test temperature:
Not applicable (calculated data)
pH:
Not applicable (calculated data)
Dissolved oxygen:
Not applicable (calculated data)
Salinity:
Not applicable (calculated data)
Nominal and measured concentrations:
Not applicable (calculated data)
Details on test conditions:
Not applicable (calculated data)
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
No details (calculated data) - See inforamtion on substance composition in "Overall Remarks" section
Results with reference substance (positive control):
No reference substance required
Reported statistics and error estimates:
Not applicable

No other information

Validity criteria fulfilled:
yes
Conclusions:
Results of computer modelling to estimate chronicity in a 21-day freshwater invertebrate study, based on reproduction, show that this substance will not produce toxicity at or below its maximum attainable water solubility
Executive summary:

The aquatic toxicity of was estimated using the Petrotox computer model, which combines a partitioning model used to calculate the aqueous concentration of hydrocarbon components as a function of substance loading with the Target Lipid Model used to calculate acute and chronic toxicity of non-polar narcotic chemicals. Petrotox computes toxicity based on the summation of the aqueous-phase concentrations of hydrocarbon block(s) that represent a hydrocarbon substance and membrane-water partitioning coefficients (KMW) that describe the partitioning of the hydrocarbons between the water and organism. Results of computer modelling to estimate chronicity in a 21-day freshwater invertebrate study, based on reproduction, show that this substance will not produce toxicity at or below its maximum attainable water solubility.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June to July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentration and stability of the test material in the treatment solutions were verified by chemical analysis of the fresh media on days 0, 1, 5, 8, 12, 15, 19, and 20, and the old media on days 1, 2, 6, 9, 13, 16, 20, and 21.
Vehicle:
no
Details on test solutions:
The test material was prepared using an aqueous vapor saturation system to provide a saturated solution. An amount of test material (75.00 g) was added to a 150 ml glass dreschel bottle (the height of the test material in the bottle was marked so that the test material could be replenished for further saturated solution preparations throughout the test) and air passed at a rate of approximately 50 cc/min through 10 L of reconstituted water for approximately 48 hours to give a saturated solution of approximately 0.04 mg/l. The remainder of the treatment solutions was prepared by serial dilutions from this saturated solution. After the preparation of one saturated solution, the dreschel bottle containing the test material was transferred to the next preparation of a saturated solution. The dreschel bottle was also topped up to the mark initially made on the bottle. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test material in the treatment solutions were verified by chemical analysis of the fresh media on days 0, 1, 5, 8, 12, 15, 19, and 20, and the old media on days 1, 2, 6, 9, 13, 16, 20, and 21.
Test organisms (species):
Daphnia magna
Details on test organisms:
Organisms used in the test were 1st instar D. magna from an in-house laboratory culture. Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water at a temperature of approximately 21 degrees C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
246 to 258 mg/L CaCO3
Test temperature:
21.3 to 22.2 degrees C
pH:
7.9 to 8.5
Dissolved oxygen:
96 to 100 % of air saturation value
Nominal and measured concentrations:
The nominal values were 0.0025, 0.0050, 0.010, 0.020, and 0.040 mg/l, which measured 0.00140, 0.00295, 0.00662, 0.0125, and 0.0291 mg/L, respectively.
Details on test conditions:
Based on the results of a preliminary range-finding test, preliminary solubility trials, the following nominal treatment levels were assigned to the definitive test: 0.0025, 0.0050, 0.010, 0.020, and 0.040 mg/l. For each treatment a single daphnid was placed in 100 ml of the test preparation in 100 ml glass flasks which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The flasks were maintained at 21.3 to 22.2 degrees C with a photoperiod of 16 hours light (647 to 701 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. Each vessel was randomly assigned to a position in the laboratory. The test vessels were not aerated. The diluent water only was aerated prior to use. The control group was maintained under identical conditions but not exposed to the test material. The test preparations were renewed daily as preliminary investigational work showed the test material to be unstable in test medium and that losses due to volatility were likely to occur. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded. Each daphnid received approximately 5.5 to 9.5 ul of a unicellular algal culture (Chlorella sp.), daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid. On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls. The number of Daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia that were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks. At the end of the test, the length of each surviving parent animal was determined.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
0.029 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: 95% CI = 0.027 to 0.032 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.02 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: 95% CI = 0.017 to 0.022 mg/L
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
0.045 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CI = 0.029 to 0.069 mg/L;
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.033 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CI = 0.020 to 0.052 mg/L
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
0.04 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobility and reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.029 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: immobility and reproduction
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
0.02 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobility and reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.013 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: immobility and reproduction
Details on results:
Immobilisation occurred predominantly at the highest test concentration of 0.040 mg/l resulting in 90% immobility by day 21. Immobility was also observed at the test concentrations of 0.0050 mg/l on day 6, 0.020 mg/l on Day 10 and 0.010 mg/l on day 18. However, statistical analysis of the immobility data using the corrected chi-squared statistic showed that the observed immobility in the 0.0050, 0.010 and 0.020 mg/l test groups was not significantly different (P≥0.05) when compared to the control group. No immobility occurred at the 0.0025 mg/l test concentration throughout the test.

There was no significant effect on the size and color of the daphnids at the 0.040 mg/l test concentration prior to observing 90% immobility by day 21. The daphnids at the remaining test concentrations were observed to be the same size and color as the control animals. After 21 days there were no statistically significant differences between the control and the 0.0025, 0.0050, 0.010, and 0.020 mg/l test groups in terms of the number of live young produced per adult. The 0.040 mg/l test group data was not included in the statistical analysis due to statistically significant mortalities in the parental (P1) generation over the duration of the test. The EC50 (reproduction) value calculated by maximum-likelihood probit method on Day 21, based on nominal test concentrations, was 0.045 mg/l with 95% confidence limits of 0.029 to 0.69 mg/l. Care should be taken in the interpretation of this EC50 value as it is in excess of the nominal concentration for the saturated solution. However analysis of the saturated solutions throughout the test showed measured concentrations to be up to 0.0534 mg/l. Therefore it was considered justifiable to calculate this value based on nominal values.

Information on the effects of the test material on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test. Young were first produced in the control group on day 7 of the test. Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

The Lowest Observed Effect Concentration (LOEC) was considered to be 0.040 mg/l on the basis that at this concentration significant immobility was observed in the parental generation (P1) and that there were significant differences (P≥0.05) between the control and the 0.040 mg/l test group in terms of numbers of live young produced per adult by day 21.

The "No Observed Effect Concentration" (NOEC) was 0.020 mg/l as there was no significant immobility observed in the parental generation (P1) and there were no significant differences (P≥0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.
Reported statistics and error estimates:
The EC50 (immobilisation) values were calculated by the trimmed Spearman-Karber method. The EC50 (reproduction) values were calculated by the maximum-likelihood probit method.
Validity criteria fulfilled:
yes
Conclusions:
The 21-Day EL50 (immobilisation) values, based on nominal values, for the parental Daphnia generation (P1) was calculated as 0.029 mg/l with 95% confidence limits of 0.027 to 0.032 mg/l. The 21-Day EL50 (reproduction) value based on nominal values was calculated as 0.045 mg/l with 95% confidence limits of 0.029 to 0.69 mg/l.
The "Lowest Observed Effect Loading Rate" was calculated as 0.040 mg/l on the basis that at this test concentration significant immobility was observed in the parental generation (P1) and that there were significant differences (P>0.05) between the control and the 0.040 mg/l test group in terms of numbers of live young produced per adult by Day 21.
The "No Observed Effect Loading Rate" was calculated as 0.020 mg/l on the basis that there was no significant immobility observed in the parental generation (P1) and that there were no significant differences (P≥0.05) between the control and the 0.020 mg/l test group in terms of numbers of live young produced per adult by Day 21.
The 21-Day EC50 (immobilisation) values based on the time-weighted mean measured test concentrations of the test media, for the parental Daphnia generation (P1) were calculated as 0.020 mg/l with 95% confidence limits of 0.017 to 0.022 mg/l.
The 21-Day EC50 (reproduction) value based on the time-weighted mean measured test concentrations of the test media was calculated as 0.033 mg/l with 95% confidence limits of 0.020 to 0.52 mg/l.
The “Lowest-Observed Effect Concentration” and the “No Observed Effect Concentration” based on the time-weighted mean measured test concentrations were 0.029 and 0.013 mg/l, respectively.
Executive summary:

The 21-Day EL50 (immobilisation) values, based on nominal values, for the parental Daphnia generation (P1) was calculated as 0.029 mg/l with 95% confidence limits of 0.027 to 0.032 mg/l. The 21-Day EL50 (reproduction) value based on nominal values was calculated as 0.045 mg/l with 95% confidence limits of 0.029 to 0.69 mg/l. Care should be taken in the interpretation of this EL50 value as it is in excess of the nominal concentration for the saturated solution. However analysis of the saturated solutions throughout the test showed measured concentrations to be up to 0.0534 mg/l. Therefore it was considered justifiable to calculate this value based on nominal values. The "Lowest Observed Effect Loading Rate" was calculated as 0.040 mg/l on the basis that at this test concentration significant immobility was observed in the parental generation (P1) and that there were significant differences (P>0.05) between the control and the 0.040 mg/l test group in terms of numbers of live young produced per adult by Day 21. The "No Observed Effect Loading Rate" was calculated as 0.020 mg/l on the basis that there were no significant immobility observed in the parental generation (P1) and that there were no significant differences (P≥0.05) between the control and the 0.020 mg/l test group in terms of numbers of live young produced per adult by Day 21. Given that the test material was shown to be unstable in test medium and that losses could be expected due to volatility, the definitive test was performed with analysis of freshly prepared and 24-hour old test media. The concentration and stability of the test material in the treatment solutions were verified by chemical analysis of the fresh media on days 0, 1, 5, 8, 12, 15, 19, and 20, and the old media on days 1, 2, 6, 9, 13, 16, 20, and 21. Analysis of the freshly prepared saturated solution showed measured test concentrations to range from 0.0233 to 0.0534 mg/l. Analysis of the freshly prepared test concentrations prepared by dilution from the saturated solution showed measured concentrations to range from <LOQ to 0.077 mg/l. Analysis of the 24-hour old test media showed the measured test concentrations to range from <LOQ to 0.0325 mg/l. Overall inspection of the data indicated a decline in measured test concentrations over the 24-hour media renewal period. This decline was considered to be due to instability of the test material in test medium and possible losses due to volatility, despite efforts being made to minimize losses by this route. The decline in measured concentrations was inline with the stability analysis performed. Given the decline in measured test concentrations it was considered justifiable to calculate the results based on the time-weighted mean measured test concentrations. The 21-Day EC50 (immobilisation) values based on the time-weighted mean measured test concentrations of the test media, for the parental Daphnia generation (P1) were calculated as 0.020 mg/l with 95% confidence limits of 0.017 to 0.022 mg/l. The 21-Day EC50 (reproduction) value based on the time-weighted mean measured test concentrations of the test media was calculated as 0.033 mg/l with 95% confidence limits of 0.020 to 0.52 mg/l. The “Lowest-Observed Effect Concentration” and the “No Observed Effect Concentration” based on the time-weighted mean measured test concentrations were 0.029 and 0.013 mg/l, respectively.

Description of key information

The test substance is poorly soluble and made of constituents with various water solubilities. As a consequence, exposures were performed with Water Accomodated Fractions (WAFs). Therefore, the results are based on nominal loadings and no NOEC value can be given.

Results of computer modelling to estimate chronicity in a 21-day freshwater invertebrate study, based on reproduction, show that this substance will not produce toxicity at or below its maximum attainable water solubility.

Key value for chemical safety assessment

Additional information

The aquatic toxicity of was estimated using the Petrotox computer model, which combines a partitioning model used to calculate the aqueous concentration of hydrocarbon components as a function of substance loading with the Target Lipid Model used to calculate acute and chronic toxicity of non-polar narcotic chemicals. Petrotox computes toxicity based on the summation of the aqueous-phase concentrations of hydrocarbon block(s) that represent a hydrocarbon substance and membrane-water partitioning coefficients (KMW) that describe the partitioning of the hydrocarbons between the water and organism. Results of computer modelling to estimate chronicity in a 21-day freshwater invertebrate study, based on reproduction, show that this substance will not produce toxicity at or below its maximum attainable water solubility.

Morover, two studies on long term toxicity to aquatic invertebrates were available and confirm the calculated result from Petrotox.. One study, dealing with the toxicity of "Hydrocarbons, C14 -C17, n-alkanes, <2% aromatics" on Ceriodaphnia dubia was considered unassignable because the concentrations were expressed only as "% WAF" (WAF = 1 V substance + 10 V dilution water). However, the NOELR was 100% WAF. A second study with Ceriodaphnia dubia exposed to "Hydrocarbons, C14-C17, n-alkanes, <2% aromatics" found that no toxic effect was obtained with 100% WAF corresponding to 70 mg/L (Dufresne, 2001b).