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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:
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:
experimental study
Adequacy of study:
supporting 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
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
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:
disregarded due to major methodological deficiencies
Study period:
February to March 2004
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This robust summary has a reliability rating of 3 because the study usied gas phase loading or Water Soluble Fraction (WSF) testing, which are not considered appropriate due to methodological issues which will impact hydrocarbon exposure concentrations.
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
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.

Description of key information

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.

Key value for chemical safety assessment

Additional information

The read across test substance C11 -C13, isoalkanes, cyclics, 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.

In a second 21-day study, the adult daphnids exposed to hydrocarbons, C10-C12, isoalkanes, <2% aromatics, 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 reported as <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. The data from these two studies are used as read-across data to hydrocarbons, C10-C13, isoalkanes, cyclics, <2% aromatics. Here we use the data from C11 -C13 isoalkanes, cyclics as our key study and therefore the apporpriate NOELR because it more closely resembles the composition of the taget substance than does the C10 -C12 isoalkanes which does not contain C13 compounds.