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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Adopted according to OECD SIDS (public available peer reviewed source) and respective study summary. The original source is not available and has not been reviewed.
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)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples were collected from each treatment level at the beginning of the test and from newly-prepared test solutions before the solutions were poured into replicate test chambers on every Friday during the test. A sample of each solution was also collected from two replicates of each treatment each Monday before media renewal and at the end of the test. If complete mortality occurred in a treatment on a Wednesday or Friday, an additional set of test solution samples was collected from that treatment on the day of complete mortality. The two test vessels from which samples were collected were selected a priori using a random numbers table and replicate samples of old solutions from each concentration were not pooled prior to analysis.

Samples (20 mL) were transferred directly into 40 mL amber glass vials containing 10 mL hexane, shaken for approximately 2 minutes, and allowed to settle until 2 layers formed. An aliquot of the top hexane layer was transferred to an amber glass autosampler vial and analyzed by GC-FID (Hewlett Packard 5890 Series II Plus).
Vehicle:
no
Details on test solutions:
Each of the five WAFs was prepared by combining the appropriate amount of test substance (0.0040, 0.0080, 0.0160, 0.0320, and 0.0400 g) and 4.0 L of dilution water in a 4 L amber glass bottle (34 cm high, including neck, and 13.5 cm in diameter at the test solution surface), equipped with a magnetic stirrer (stir bar length = 50 mm and stir bar diameter = 10 mm). The control consisted of 4.0 L of dilution water and no test substance.

The mixing vessels had a spigot (approximately 7 mm diameter) located approximately 10 mm from the bottom. The test substance was added to the dilution water before stirring began and stirring was then adjusted so that the vortex caused only a slight dimple on the water surface. The vortex depth was approximately 10 mm and the test solution height in the mixing vessels was approximately 24 cm at the beginning and end of each stirring period. At the end of the 24 hour stirring period, the solutions were allowed to stand for 4 hours and then each WAF was gently and quickly removed through the spigot from the mixing vessel into test vessels. During the definitive toxicity test a subsample of each WAF was checked microscopically for the presence of micro-dispersions/droplets of the test substance, and none were found.

The stirring time needed to reach equilibrium was determined experimentally using the same type of glassware that was used in the toxicity test. Two identical 10 mg/L WAFs were prepared and mixed as described in the preceding paragraphs. The first WAF, which was sampled after 24 hours of mixing, had a measured concentration of 1.50 mg/L of test substance and the second WAF, which was sampled after 48 hours of mixing, had a measured concentration of 1.53 mg/L. Based on this experiment, the mixing interval was set at 24 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Feeding during test
- Food type: Daphnids were fed the freshwater alga, Selenastrum capricornutum, and a yeast/trout chow mixture
- Amount: approximately 0.01 mg carbon/daphnid/day until day 14 and then 0.1 to 0.2 mg carbon/daphnid/day
- Frequency: at each media renewal; The feeding rate was adjusted to insure adequate food for growth and reproduction while minimizing alterations of water quality in the sealed vessels
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Test temperature:
20 ± 1°C
Dissolved oxygen:
¿ 5.8 mg/L, or approximately 64% saturation in the sealed test vessels (saturation equals 9.3 mg/L at 19°C to 8.9 mg/L at 21°C)
Nominal and measured concentrations:
0, 1.0, 2.0, 4.0, 8.0, and 10 mg/L (nominal)
none detected, 0.17, 0.32, 0.79, 1.1, and 1.2 mg/L (mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 120 mL clear glass jars that were filled to capacity and closed with Teflon-lined caps, eliminating all head space (water depth was approximately 7 cm)
- Renewal rate of test solution (frequency/flow rate): Test organisms were transferred to freshly prepared test solutions every Monday, Wednesday, and Friday during the toxicity test and test vessels remained tightly sealed between media renewals.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light and 8-hour dark photoperiod, a 15-minute transition period was provided between dark and light
- Light intensity: cool white fluorescent lights that provided a light intensity of approximately 5 µEin/m2sec


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Survival of first generation daphnids, the average number of young per daphnid, the time to first brood, and the length of surviving first generation daphnids were statistically analyzed.
The number of surviving adult daphnids and the occurrence of sublethal effects (immobilization) were determined every day of media renewal during the test. Dead test organisms were removed at these times. The number of young produced by the daphnids in each test vessel were determined and recorded on every day of media renewal after the onset of reproduction, and the young were removed from each test vessel at these times. At the termination of the test,adult survivors were measured (body length excluding the anal spine).


RANGE-FINDING STUDY
- Test loadings: 0, 1.0, 10, 50, 100, and 1,000 mg/L
- Results used to determine the conditions for the definitive study: After 14 days, there was 100% survival in the control and at 1.0 mg/L, and 0% survival at 10, 50, 100, and 1,000 mg/L. Young production averaged 1.8 per female in the control and 5.6 per female at 1.0 mg/L. A definitive test was conducted with a control and five nominal loading levels of the WAF (1.0, 2.0, 4.0, 8.0, and 10 mg/L).
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
not specified
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
not specified
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
not specified
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
not specified
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
not specified
Remarks on result:
other: 95% CL 2.0 to 8.0 mg/L
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
not specified
Remarks on result:
other: 95% CL 1.0 to 4.0 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.64 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
not specified
Remarks on result:
other: 95% CL 0.32 to 1.1 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Water Accomodated Fraction (WAF)
Basis for effect:
not specified
Remarks on result:
other: 95% CL 0.17 to 0.32 mg/L
Details on results:
There was 100% control survival 21 days after test initiation. Production of young by the first generation daphnids was first observed on day 12 in the control and first generation control daphnids produced an average of 85 young during the 21 day exposure (CV = 15%). At the end of the test the control daphnids had an average length of 3.8 mm.
No mortality, sublethal effects, or reproductive or size reductions were observed at a nominal loading of 1.0 mg/L during the test. The survival and length of daphnids was significantly reduced at 2.0 mg/L after 21 days of exposure and no offspring production occurred. No survival of daphnids occurred at nominal loadings above 2.0 mg/L. No immobilized young, or dead eggs were observed during the test.
Reported statistics and error estimates:
Results of the toxicity test were analyzed, when warranted, by standard statistical techniques using TOXSTAT version 3.3 (Gulley et al., 1990). A chi square test was used to determine if data were normally distributed and Bartlett's test was used to determine if variances were homogeneous. When data were normally distributed and variances were homogeneous, a one-way analysis of variance (ANOVA) and Dunnett¿s test were used to compare treatment data to control data. When data were not normally distributed or variances were not homogeneous, a nonparametric William¿s test was used to compare treatment data to control data.

All measured biological endpoints (survival, sublethal effects, reproduction, and length of first generation adults) are equally sensitive when comparisons are made between the treatments and the control.

12 to 20% of the nominal loading rate was dissolved in the water phase of the WAFs used during the toxicity test. The mean measured concentrations in freshly prepared test solutions (day 0, 7, 14, and 19 samples) averaged 12 to 22% of nominal loading rates and these concentrations were consistent from batch to batch for each concentration. Lower relative dissolved concentrations in the freshly prepared WAFs at the two highest test concentrations (13 and 16% vs. 21 to 22% at the three lowest concentrations) suggest that 1.2 mg/L might be the saturation limit under test conditions.

Tab.: Endpoint results for the control and test solutions

 

Nominal Loading Level of Test Substance (mg/L)

Biological Endpoint

Control

1.0

2.0

4.0

8.0

10

Percent Survival at Day 21

100

100

20*

0**

0**

0**

Immobilization at Day 21

none

none

none

--**

--**

--**

Mean Day of First Brood

13.8

13.2

>21*

--**

--**

--**

Mean Young per Surviving Daphnid

85

85

0*

--**

--**

--**

Mean Length of Surviving Adults (mm)

3.8

3.9

2.8*

--**

--**

--**

*  statistically different than the control (P<0.05)

** assumed to be significantly different than the control because no

    first generation control daphnids survived

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study a NOELR and an EL50 of 1 and 1.6 mg/L, respectively, were determined for the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics.
Executive summary:

Under the conditions of the study a NOELR and an EL50 of 1 and 1.6 mg/L, respectively, were determined for the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics.

Description of key information

NOELR (21 d) = 1 mg/L (reproduction) (Daphnia)

Key value for chemical safety assessment

Additional information

No studies are available on the chronic toxicity to aquatic invertebrates for Hydrocarbons, C6-C10 (even numbered), n-alkanes, isoalkanes, >5% n-hexane. Based on a category approach, data are read-across to hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics. A study conducted according to OECD 211 guideline is available on the chronic toxicity to Daphnia for hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics (Wilbury Laboratories 2004). The chronic toxicity, as measured by survival, sublethal effects, reproduction, and length of first generation adults, was evaluated over a period of 21 days in freshwater by using water accomodated fractions (WAFs). Under the conditions of this study, the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, produced chronic toxicity to Daphnia at 1.6 mg/L based on nominal loadings of the test substance in water. A NOELR was determined to be 1 mg/L.

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