Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2000-04-04 to 2000-06-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with agreed test protocols following OECD Method 211. Acceptable restriction was that analytical measurements on the test material were not conducted.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
2000-04-26

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: amber viscous liquid
- Physical state: liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in the dark
- Other: none
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
none.

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations: 1.5, 4.8, 15, 48, and 150 mg/L
- Sampling method: The concentration and stability of the test material in the test solutions were verified by chemical analysis on days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 and 21 (old media)

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Loading rates
Based on the results of an acute toxicity test to 1st instar Daphnia magna (Safepharm Laboratories Project Number 525/259) the following nominal loading rates were assigned to the study: 1.5, 4.8, 15, 48 and 150 mg/l.
- Method:A WAF of nominal loading rate of 1000 mg/L was prepared, in duplicate, in reconstituted water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1 hour standing period each of the mixtures was filtered through 0.2 µm filters and the concentration of the test material in the filtered WAFs verified by Dissolved Organic Carbon (DOC) analysis. Filtration was conducted as undissolved test material was observed throughout the water columns of the test loading rates at the end of each preparation period during WAF method development work. It was considered that careful siphoning of the loading rates would not have removed all of the undissolved test material as it may have exhibited a physical effect on the test organisms during testing.
- Eluate: Reconstituted water.
- Differential loading: Amounts of test material (15, 48, 150, 480, 1500 mg) were each separately added to the vortex created in 10 L of reconstituted water to give the 1.5, 4.8, 15, 48, and 150 mg/L loading rates.
- Controls: The control group was maintained under identical conditions but not exposed to the test material.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnids
- Strain/clone: 1st instar
- Source: in-house laboratory
- Age of parental stock (mean and range, SD): no data
- Feeding during test
- Food type: Chlorella sp.
- Amount: 30 x 10^9 cells per day
- Frequency: daily

ACCLIMATION
- Acclimation period: no data
- Acclimation conditions (same as test or not): Adults were maintained in polypropylene vessels containing approximately 2 L of reconstituted water at 21°C.
- Type and amount of food: Chlorella sp. 30 x 10^9 cells per day
- Feeding frequency: daily

On a daily basis, the numbers of live and dead of the parental generation, the numbers of live and dead filial 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 at each meadia renewal. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 sec after gentle agitation, 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 study, the length of each surviving parent animal was determined.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none.

Test conditions

Hardness:
The water hardness was observed to be in the range 218 to 256 mg/l as CaCO3 in the control and the highest surviving test group throughout the study.
Test temperature:
21.0°C
pH:
7.7-8.0
Dissolved oxygen:
8.0-8.4 mg O2/L
Salinity:
not applicable.
Nominal and measured concentrations:
nominal concentrations: 0, 1.5, 4.8, 15, 48, 150 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 150 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 3 times per week (days 2, 5, 7, 9, 12, 14, 16 and 19).
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 light:8 dark
- Light intensity: ca. 132 - 152 lux.

Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
14 d
Dose descriptor:
EL50
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: 33-69
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
27 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: 15-48
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 27-37
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Details on results:
none
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
none.

Any other information on results incl. tables

Study data are presented in attached document (cf. Tables.pdf)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 14 and 21 -day EL50 (immobilisation) values, based on the nominal loading rates, for the parental Daphnia generation were calculated to be 48 and 27 mg/L loading rate filtered WAF with 95% confidence limits of 33 -69 mg/L loading rate filtered WAF and 15 -48 mg/L loading rate filtered WAF respectively. The 21 -day EL50 (reproduction) was calculated to be 31 mg/L loading rate filtered WAF with 95% confidence limits of 37 -37 mg/L. The LOEC is considered to be 48 mg/L loading rate filtered WAF on the basis that at this loading there were significant mortalities (immobilisation) observed in the parental generation. The NOEC is considered to be 15 mg/L loading rate filtered WAF on the basis that at this loading rate there were no mortalities (immobilisation) observed in the parental generation and that there were no significant differneces between the control and the 15 mg/L loading rate filtered WAF test group in terms of the number of live young produced per adult by day 21.
Executive summary:

Methods: A study was performed to assess the effect of the test material on the reproduction of Daphnia magna over a 21 day period. The method followed that described in the OECD Guidelines No 211 "Daphnia magna, Reproduction test".

Procedure: Based on the results of an acute toxicity study, Daphnia magna were exposed (10 replicates of a single daphnid per group) to filtered WAF of the test material over a range of nominal loading rates of 1.5, 4.8, 15, 48, 150 mg/L for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids were determined daily. The Daphnia were fed daily with an algal suspension.

Results: Analysis of the test loading rates at 0 hours showed the measured concentrations to range from 0.106 to 0.826 mg/L whilst analysis of the old or expired media sampled throughout the study showed measured concentrations to range from less than the limit of quantification of the analytical method to 0.863 mg/L. These results were in-line with the recovery and stability analyses performed on the WAFs which showed the test material to be unstable in both light and dark conditions at the lower test concentrations. On the majority of sampling occasions increasing measured concentrations were shown with increasing loading rates.

Given that the toxicity cannot be attributed to any one or more of the components in the WAF, but to the mixture as a whole, the results are based on nominal loading rates only.

The 14 and 21 -day EL50 (immobilisation) values, based on the nominal loading rates, for the parental Daphnia generation were calculated to be 48 and 27 mg/L loading rate filtered WAF with 95% confidence limits of 33 -69 mg/L loading rate filtered WAF and 15 -48 mg/L loading rate filtered WAF respectively.

The 21 -day EL50 (reproduction) was calculated to be 31 mg/L loading rate filtered WAF with 95% confidence limits of 37 -37 mg/L.

The LOEC is considered to be 48 mg/L loading rate filtered WAF on the basis that at this loading there were significant mortalities (immobilisation) observed in the parental generation.

The NOEC is considered to be 15 mg/L loading rate filtered WAF on the basis that at this loading rate there were no mortalities (immobilisation) observed in the parental generation and that there were no significant differences between the control and the 15 mg/L loading rate filtered WAF test group in terms of the number of live young produced per adult by day 21.