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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)

Data source

Reference Type:
The acute and chronic toxicity of two xanthene dyes, fluorescein sodium salt and phloxine B, to Daphnia pulex
W.K. Walthall, J.D. Stark
Bibliographic source:
Environmental Pollution 104 (1999) 207-215

Materials and methods

Principles of method if other than guideline:
Long term toxicity to aquatic invertebrates study was performed to evaluate the chronic toxic nature of the test compound Fluorescein sodium
GLP compliance:
not specified

Test material

Test material form:
other: Solid
Details on test material:
- Name of test material (as cited in study report): C20-H10-O5.2Na
- Molecular weight (if other than submission substance): 376.274 g/mol
- Smiles notation (if other than submission substance): c1(c2c(oc3c1ccc(c3)[O-])cc(=O)cc2)c1c(cccc1)C(=O)[O-].[Na+].[Na+]
- InChl (if other than submission substance): 1S/C20H12O5.2Na/c21-11-5-7-15-17(9-11)25-18-10-12(22)6-8-16(18)19(15)13-3-1-2-4-14(13)20(23)24;;/h1-10,21H,(H,23,24);;/q;2*+1/p-2
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data

Sampling and analysis

Analytical monitoring:
not specified
Details on sampling:
0, 200, 250, 350, 450, and 600 mg/liter
- Sampling method: All sample concentrations were prepared by serial dilution from a freshly prepared stock solution. Stock solutions were prepared by the addition of a measured dye sample in a defined volume of RDW.

- Sample storage conditions before analysis: Test containers were placed on a styrofoam tray, covered with a piece of plexiglass to reduce evaporation, and held in an environmental chamber set as previously described.

Test solutions

not specified
Details on test solutions:
- Method: At each concentration, 100ml of test solution, prepared by serial dilution from a freshly prepared stock solution, was transferred into a 296-ml (10 oz.) SOLO1 Ultra Clear2 plastic cup. Control chambers contained 100 ml RDW only. Ten neonates were transferred into each test container using a disposable glass pipette.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
All sample concentrations were prepared by serial dilution from a freshly prepared stock solution. Stock solutions were prepared by the addition of a measured dye sample in a defined volume of RDW. Each test consisted of a minimum of five log-spaced concentrations and a RDW control

Test organisms

Test organisms (species):
Daphnia pulex
Details on test organisms:
- Common name: Daphnia pulex
- Strain: No data
- Source: Washington State University Research and Extension Center
- Age at study initiation (mean and range, SD): (<24 h old)

- Valve height at study initiation, for shell deposition study (mean and range, SD):
- Peripheral shell growth removed prior to test initiation:
- Method of breeding:
- Feeding during test:
- Food type: The food solution consisted of a 1:1 mixture of a yeast/cereal leaves/trout chow (YCT) solution and the green algae species Selenestrum capri- cornutum.
- Amount: No data
- Frequency: Test organisms were allowed to feed for a minimum of 2 h prior to introduction to the sample solution.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
10 d
Post exposure observation period:
At 10 days post-introduction, test populations were removed from the environmental chamber and the population in each cup was censused. Day 10 was chosen as the time to census because it was the minimum time necessary to guarantee a minimum of two broods for individuals in the control populations.

Test conditions

80-100 mg/liter.
Test temperature:
Dissolved oxygen:
No data
No data
Nominal and measured concentrations:
No data
Details on test conditions:
- Range finding study: Yes
- Test concentrations for range finding study: 0, 150, 250, 400, 600 and 800 mg/liter for range finding study
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
not specified

Results and discussion

Effect concentrations
10 d
Dose descriptor:
other: LC90
Effect conc.:
721 mg/L
Conc. based on:
test mat.
Details on results:
No data

Any other information on results incl. tables

At a concentration of 600 mg/liter, a concentration corresponding to the LC83, all individuals were dead following 10-day exposure.

Applicant's summary and conclusion

Fluorescein appeared to elicit a sublethal effect on the reproductive potential of surviving individuals. The LC90 was found to be 721 mg/litre for chronic study.
Executive summary:

The toxicity of dye, fluorescein sodium salt, to Daphnia pulex was assessed using chronic level toxicological study for 10days. Chronic toxicity experiments were initiated with neo-nates (<24 h old) removed from cultures and allowed to feed in a 150-ml beaker with 3 ml of food solution for a minimum of 2 h prior to introduction to the sample solutions. Preliminary tests were conducted to determine the range of concentrations necessary to elicit a sublethal response. Based on these preliminary tests the series of concentration used are 0, 200, 250, 350, 450, and 600 mg/liter. Fluorescein elicits greater sublethal effects on D. pulex neonates. The reproductive potential of the censused control populations significantly (p<0.05) exceeded that of all other tested concentrations, with increasing concentration resulting in a concentration-dependent reduction in the mean number of offspring per surviving female. This was most apparent at 350 mg/liter where mean survivorship was 80%, but the mean reproductive potential was 4.65 offspring per surviving female compared to 25 offspring per surviving female in the control. Following 10-day exposure under static conditions, D. pulex neonates exposed to concentrations of fluorescein at 250 mg/liter and below did not show large declines in survivorship; however, exposure at these concentrations did result in substantial declines in the reproductive potential of surviving individuals. The concentration of fluorescein necessary to elicit even a sublethal response was quite high. Based on the results the LC90 was found to be 721 mg/litre for chronic study.